Tree Physiology Advance Access published October 7, 2016
Tree Physiology, 1–10
doi:10.1093/treephys/tpw092
Research paper
Significant contribution from foliage-derived ABA in regulating gas
exchange in Pinus radiata
1
3
CSIRO Land and Water, College Rd, Sandy Bay, Tasmania 7005, Australia; 2School of Biological Sciences, University of Tasmania, College Rd, Hobart, Tasmania 7005, Australia;
Corresponding author ([email protected])
Received April 21, 2016; accepted August 20, 2016; handling Editor Frederick Meinzer
The complex regulatory system controlling stomata involves physical and chemical signals that affect guard cell turgor to bring
about changes in stomatal conductance (gs). Abscisic acid (ABA) closes stomata, yet the mechanisms controlling foliar ABA status
in tree species remain unclear. The importance of foliage-derived ABA in regulating gas exchange was evaluated under treatments that affected phloem export through girdling and reduced water availability in the tree species, Pinus radiata (D. Don).
Branch- and whole-plant girdling increased foliar ABA levels leading to declines in gs, despite no change in plant water status.
Changes in gs were largely independent of the more transient increases in foliar non-structural carbohydrates (NSC), suggesting
that gradual accumulation of foliar ABA was the primary mechanism for reductions in gs and assimilation. Whole-plant girdling
eventually reduced root NSC, hindering root water uptake and decreasing foliar water potential, causing a dramatic increase in
ABA level in leaves and concentrations in the xylem sap of shoots (4032 ng ml−1), while root xylem sap concentrations remained
low (43 ng ml−1). Contrastingly, the drought treatment caused similar increases in xylem sap ABA in both roots and shoots, suggesting that declines in water potential result in relatively consistent changes in ABA along the hydraulic pathway. ABA levels in
plant canopies can be regulated independently of changes in root water status triggered by changes by both phloem export and
foliar water status.
Keywords: ABA, drought, girdling, non-structural carbohydrates, phloem transport, stomatal conductance..
Introduction
By changing stomatal aperture to regulate the exchange of water
vapour and CO2, plants can respond dynamically and rapidly to
fluctuations in environmental conditions and maximize efficiencies in water use. Exposure to water deficit during conditions of
high evaporative demand or a decline in root zone water availability can reduce stomatal conductance (gs) and transpiration.
Stomatal conductance also responds to variation in carbon
source:sink strength that can arise from changes in the supply of
CO2, growth, phenology or canopy damage (Ainsworth and
Rogers 2007, Pinkard et al. 2011). The complex regulatory system controlling stomata involves physical and chemical signals
(Comstock 2002) that affect guard cell turgor to bring about
changes in stomatal conductance (gs).
In seed plants, the phytohormone abscisic acid (ABA) acts as
an inhibitor of gs by mediating guard cell turgor through changes
in the activity of membrane channels and transporters (Assmann
and Shimazaki 1999, Geiger et al. 2009). Enzymes involved in
the biosynthesis of ABA are expressed in the vascular tissues of
roots, shoots and foliage (Cheng et al. 2002) in response to
stress such as water deficit (Endo et al. 2008), and this
vascular-derived ABA primarily contributes to stomatal closure
via transport to the guard cells (Seo and Koshiba 2011,
Kuromori et al. 2014). The perception of water shortages by
root tips is thought to stimulate ABA synthesis that is transported
to the shoot via the transpiration stream to provide an ‘early
warning’ stress signal to the canopy (Wilkinson and Davies
2002). Evidence for long-distance signalling from the roots
© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
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Patrick J. Mitchell1,3, Scott A. M. McAdam2, Elizabeth A. Pinkard1 and Timothy J. Brodribb2
2 Mitchell et al.
Tree Physiology Volume 00, 2016
efficacy of a long-distance chemical signalling in regulating tree
gas exchange and the limited evidence demonstrating a close
relationship between levels of ABA in the xylem sap and gs in the
field have focused forest models towards hydraulic-based frameworks (Sperry 2000). These models have been used to predict
the highly dynamic responses of tree canopies to diurnal and
sub-daily changes in water status such as the depression in midday transpiration in response to fluctuating atmospheric demand
under static soil moisture contents (Gao et al. 2005). Recent
studies have demonstrated an important role for both hydraulic
and active signalling, mediated by ABA, in determining plant gas
exchange, with the relative contribution of both modes of stomatal control varying among species and providing mechanistic
explanations for the isohydric or anisohydric responses to
drought stress (McAdam and Brodribb 2012, Brodribb and
McAdam 2013).
In this study, we test the hypothesis that foliage-derived ABA
is an important driver for gas exchange, using manipulation of
phloem export and water deficit to alter foliar and root ABA status. We conducted a set of stem girdling and drought experiments that aimed to address the following questions. (i) What
role do changes in foliar ABA levels have on leaf gas exchange
in response to impeded phloem export? This was addressed by
restricting phloem export under well-watered conditions. (ii) Are
foliar ABA levels sufficient to close stomata during drought in the
absence of root-sourced ABA? Water stress in the canopy was
induced in intact plants under both well-watered and low soil
water content to identify the patterns in ABA status in both the
canopy and root tissues. Pinus radiata (D. Don) was used as a
model species in this study because it is well established that
foliar ABA levels play a strong role in regulating gs evidenced by
the high sensitivity of foliar ABA levels to changes in water status
(Brodribb and McAdam 2013). Like other conifers, the
response of gs to changes in foliar ABA level could be investigated in the absence of hydropassive, wrong-way stomatal
responses to changes in leaf water status that can complicate
the interpretation of the role of ABA in mediating stomatal behaviour in angiosperms (Brodribb and McAdam 2013).
Materials and methods
Plant material
Pinus radiata saplings (~3 years old) were used for all experiments and were initially grown outside in Hobart, Tasmania
(42.88°S, 147.32°E). Plants were grown in 75 l bags containing a potting mix consisting of 8:2:1 mix of composted pine
bark, coarse river sand and peat moss. During this time, plants
received regular applications every 3 months of standard
controlled-release fertilizer (N:P:K–19.4:1.6:5). Eight weeks
prior to the commencement of the experiments, most plants
were transferred to a glasshouse and were ~1.1 m tall. Growth
conditions in the glasshouse were 16 h days (supplemented
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comes from studies involving root pressurization and split-root
experiments in herbaceous and woody plants (e.g. Davies and
Zhang 1991, Schurr et al. 1992). However, several lines of evidence suggest that stomatal regulation can occur in the absence
of a root-derived ABA source. The accumulation and synthesis of
ABA in leaves of herbaceous species in response to water deficit
has been observed using in vivo imaging (Christmann et al.
2005, Kuromori et al. 2014) and in mutants that do not synthesize ABA in roots (Holbrook et al. 2002, Manzi et al.
2015). Strong correlations between foliar ABA levels and gs
observed in whole plants in the field and controlled environmental conditions as well as detached stems exposed to water stress
are consistent with the functional pool of ABA being primarily
derived from the foliage (McAdam and Brodribb 2014). These
patterns in ABA dynamics suggest that the propagation of
hydraulic signals can provide a trigger for ABA synthesis within
vascular tissues in different organs along the water transport
pathway and may be dependent on the species responses and
the water stress conditions.
The occurrence of ABA in the phloem permits delivery of ABA
to sink tissues in the canopy and throughout the plant body, suggesting that ABA produced in foliage may be sensitive to
changes in phloem transport (Hoad 1995, Zhong et al. 1996,
Manzi et al. 2015). Studies manipulating the sink demand of
photosynthates to induce changes in plant ABA dynamics have
provided evidence that small levels of ABA synthesized in an
unstressed canopy can rapidly build-up if phloem export is
impeded, yet the functional significance of this elevated foliar
ABA level for gas exchange has not always been clear (Mich
et al. 1984, López et al. 2015). Changes in foliar ABA levels
may provide a mechanism through which changes in longdistance assimilate transport and canopy non-structural carbohydrates (NSC) are linked to rates of gas exchange (Nikinmaa
et al. 2013). For example, the transport of ABA in the phloem
can increase sucrose uptake in sink tissues (Schussler et al.
1991) and may be an important source of ABA in the xylem sap
(Neales and McLeod 1991). Manipulating phloem export by
girdling stem tissues can be used to detect attendant changes in
ABA export (and potentially synthesis) in source tissues such as
leaves and its role in mediating stomata in the absence of
changes in water status in the soil. Using this approach, the significance of changes in foliar ABA status could be distinguished
from water deficit conditions that can trigger synthesis of ABA
within potential sources from below- and above-ground tissues.
The contribution from foliage-sourced ABA for stomatal control may provide a better explanation for rapid dynamic
responses of stomata in tall trees to water deficits than a rootderived ABA source. A more localized ABA signal originating
from the foliage overcomes issues surrounding the effectiveness
of a xylem flux-mediated signal for stomatal closure that may
become heavily attenuated under moderate declines in sap flow
during water deficit. The perceived limitations regarding the
Contribution of foliar-derived ABA to gas exchange
and extended in the morning and evening by sodium vapour
lamps ensuring a minimum 300–500 μmol quanta m−2 s−1 at
the leaf surface during the day period) and 23 and 15 °C day
and night temperatures, respectively. Vapour pressure deficit
(VPD) in the glasshouse typically fluctuated from 0.8 to 1.2 kPa.
Four plants remained outside under ambient conditions.
Ambient conditions in the field ranged from 1 to 28 °C with a
mean of 12 °C and VPD ranged from 0 to 2.4 kPa with a daytime
mean of 1.1 kPa. All plants were watered daily to full soil capacity, except for individuals subjected to the drought treatment
(see below).
Girdling was applied to both the stem and multiple branches
within the canopy to compare the effect of removing all phloem
transport to roots and partial reductions in phloem transport that
likely had limited impact on root functioning. Girdling involved
removing a 15 mm band of periderm and phloem using a razor
blade. Whole-plant or stem girdling involved the removal of a
10 mm section of periderm and phloem on the main stem at
0.1 m above the soil surface in three plants. Branch girdling was
done in a similar manner on two lateral branches per tree, at
0.1 m above the stem–branch junction, in three plants growing
in the glasshouse and three plants growing outside under ambient conditions. Branch-girdled trees had a total of 8–10 lateral
branches, so that the proportion of girdled canopy was generally <30%. Neighbouring non-girdled branches were used as
controls. Silicon grease was applied to all girdled sections of
stem and any new growth of wound tissue was removed to
ensure that the phloem remained severed. Thirty-eight days after
stem girdling, plants were harvested for analysis of xylem sap
ABA concentration and NSC (see below). The drought treatment
involved withholding water from three non-girdled plants to
achieve a dry-down that lasted 34 days until harvest. For each
droughted plant, two branches were girdled, while two branches
of similar size and height were used as a non-girdled comparison. In both whole-plant girdling and drought experiments, three
well-watered plants were used as controls.
Gas exchange and leaf water status
Measurements of gas exchange and leaf water status were conducted on sunny days and were staggered so as to capture both
the initial (3–10 days after applied treatment) and protracted
(11–58 days after applied treatment) responses to both girdle
and drought treatments. The response of light-saturated photosynthesis (Asat), stomatal conductance (gs) and the CO2 partial
pressure within the leaf intercellular spaces (ci) to changes in
phloem export and water availability was quantified using an infrared gas analyser (Li6400, Licor, Lincoln, NE, USA). Conditions
inside the leaf cuvette (20 × 30 mm) were maintained to ensure
maximum needle gas exchange with VPD maintained between 1
and 1.4 kPa, CO2 concentration set at ambient, light intensity set
at 1500 µmol m−2 s−1 and block temperature set at 20 °C. A single measurement was taken on two fascicles (six needles) after
CO2 and water vapour concentrations stabilized (~1–3 min). Two
measurements per plant or branch treatment of stomatal conductance (gs) and light-saturated photosynthesis (Asat) were performed between 12:00 and 14:00 h.
Leaf water potential (Ψl) was measured using a Scholander
pressure chamber (PMS Instruments, Corvallis, OR, USA). For
the girdling and drought experiments, two fascicles were
sampled per plant. Upon removal, the fascicle was immediately
wrapped in moist paper towel and bagged until measurement in
the laboratory. Measurements of predawn leaf water potential
were conducted on needles collected between 5:00 and 6:00 h
for stem girdling and drought experiments, while daytime Ψl
were collected between 12:00 and 14:00 h in conjunction with
leaf gas exchange measurements for all experiments.
Xylem sap extraction and sapwood water content
At the end of the whole-plant girdling and drought experiments,
trees were harvested for xylem sap ABA quantification, sapwood
water content and NSC concentrations (see below). Xylem sap
for ABA analysis was extracted from an entire lateral branch (6–
10 mm diameter) or coarse root (3–6 mm diameter). Branches
or roots were removed from the plant and the distal end of
branch or root, following protocols described for needles above,
and inserted into a pressure chamber with the cut end (periderm
removed) protruding from the lid. A balance pressure was
applied to the chamber and 1–2 ml sap exuded and collected for
ABA quantification (see below) from the cut surface.
Sapwood water content was estimated at the end of the stem
girdling and drought experiments to determine the level of dehydration in xylem tissues. A 20–30 mm stem section was sampled
at 0.2 m above the soil surface, and the periderm was removed
before the determination of fresh mass. Dry mass was then determined after oven-drying the samples at 70 °C for 1 week.
Percentage sapwood water content was calculated as fresh mass
minus dry mass divided by fresh mass.
ABA extraction, purification and quantification
Foliage for ABA measurements was sampled from a neighbouring fascicle to those used for gas exchange measurements. The
leaves were removed from the plant and wrapped in moist paper
towel to prevent dehydration. Extraction, purification and
physicochemical quantification by ultra-performance liquid chromatography with an added internal standard of ABA levels were
undertaken according to the protocol of McAdam (2015).
NSC analysis
Samples for NSC analysis of foliage were taken at regular intervals during the girdling experiments and foliage and root NSC
samples were taken at the end of the whole-plant girdling and
drought experiments. Immediately after sampling, all plant
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Girdle and drought treatments
3
4 Mitchell et al.
materials were frozen at −20 °C to rapidly kill them and then
freeze-dried before being milled to a fine powder. Soluble carbohydrates were extracted as described in Mitchell et al.
(2013) using 80% ethanol and water extraction procedure.
Soluble sugars and starch concentrations (mg g−1) were calculated as content of the measured pool divided by dry mass of
the sample.
Statistical analyses
Results
Effects of impeded phloem transport
Stem girdling disrupts whole-plant phloem export dynamics, and
in P. radiata (grown in the glasshouse under constant daytime
air temperature) resulted in a steady increase in foliar ABA levels
from mean pre-girdling levels of 54–155 ng g−1 in girdled plants
after 12 days (Figure 1B). For the first 21 days, no change in
predawn Ψl from control plants occurred in the stem-girdled
plants, while midday Ψl exhibited small increases (Figure 1C and
D). Over this period, we observed a reduction in gs of up to 55%
from ~220 to 95 mmol m−2 s−1 (Figure 1A). Branch girdling
under similar glasshouse conditions caused an increase in foliar
ABA levels that peaked 2 days after girdling at 118 ng g−1 and
subsequently declined back to control levels by 50 days after
girdling (Figure 2A). For branch-girdled plants growing outside
the glasshouse, the increase in foliar ABA levels was more dramatic and continued to increase during the experiment, reaching
a maximum of 256 ng g−1, 58 days following girdling
(Figure 2B). These changes in foliar ABA levels coincided with a
~80% reduction of gs from 123 to 19 mmol m−2 s−1
(Figure 2D). Declines in gs arising from the branch girdle treatments resulted in an increase in midday Ψl (Figure 2C and F).
Under all three of the experimental treatments, there was a significant negative curvilinear relationship between gs and foliar
ABA level (R2 = 0.62–0.88; P < 0.05), over a range of ABA
levels (~30–300 ng g−1; Figures 1A, and 2A and B). Thus,
under well-watered conditions and no significant declines in Ψl
(Figures 1C and D, and 2C and F), the restriction of phloem
export from leaves leads to an increase in ABA levels in the
shoot and consistent stomatal responses at both the branch and
whole-canopy levels. Under the stem and branch girdling (field
conditions) treatments that induced large declines in gs and Asat,
internal [CO2] was also significantly reduced despite no change
in water availability (see Figure 3 for an example).
Tree Physiology Volume 00, 2016
Figure 1. Whole-tree girdling (main stem) in P. radiata results in a
decrease in stomatal conductance and an increase in foliar ABA level.
(A) Response of stomatal conductance to fluctuating levels of foliar ABA
level (log scale) in girdled (solid circles) and control plants (open circles). Data are fitted with a power function of the form y = a × xb
(P < 0.05). (B) Changes in foliar ABA level (±1 SE) through time (Day 0
represents start of treatment) in girdled (solid bar) and control (open
bar) at different sampling times. Leaf water potential (±1 SE) for (C) predawn and (D) midday measurements for four sampling times. Asterisks
denote significant difference between treatments (P < 0.05). The grey
shading denotes the period where predawn water status was lower in
girdled trees than control trees.
The stem girdling experiment showed a small and nonsignificant increase in foliar starch level, while the branchgirdling experiments induced large increases in starch level initially (at 7 and 9 days; Table 1). However, these increases in
foliar starch levels were transient and had returned to control
levels at between 35 (stem girdling) and 50 and 58 days
(branch girdling). Soluble sugar concentrations tended to show
smaller increases relative to control (Table 1). Non-structural
carbohydrates pooled across all sampling times for each experiment were only negatively correlated with gs for the branchgirdling experiment under glasshouse conditions (R2 = 0.57
and 0.54 for soluble sugars and starch, respectively; Table 1).
The impact of declining plant water status
After 35 and 33 days of the whole-plant (stem) girdle and
drought treatments, respectively, significant declines in gs (97
and 85% of controls, respectively; Figures 1A and 4A) and leaf
water status (Figure 5A) were observed under very different soil
water conditions (~100 and 23% of field capacity, respectively). Between Days 21 and 35 after stem girdling predawn Ψl
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Pair wise comparisons between treatments for foliar ABA levels,
xylem sap concentrations, foliar NSC, leaf water potential, gas
exchange variables and stem water content in each experiment
were compared using Student’s t-test (P < 0.05). All statistical
analyses were performed in Sigma Plot (v 12.5, Systat Software
Inc., San Jose, CA, USA).
Contribution of foliar-derived ABA to gas exchange
5
in girdled plants declined dramatically from −0.3 to −2.0 MPa
(Figure 5A), while foliar ABA levels increased from 126 to
1129 ng g−1 (Figure 1B). Under these conditions, xylem sap
ABA concentrations in the shoots mirrored foliar patterns; reaching 4032 ng ml−1 (versus 1129 ng g−1 in leaf tissue), whereas
the concentration of ABA in root xylem sap remained low (43 ng
ml−1) and similar to control levels (8 ng ml−1; not significant;
Figure 5C).
When water was withheld for 33 days in plants that were not
stem-girdled, a steady accumulation of foliar ABA was observed
peaking at a mean of 219 and 196 ng g−1 in non-girdled and
girdled branches, respectively, as daytime Ψl reached ~
−1.2 MPa (Figures 5 and 6). During the drought period, foliar
ABA levels were positively correlated with both predawn and
midday Ψl (R2 = 0.63–0.73, P < 0.05; Figure 6). Under
drought, xylem sap concentrations of non-girdled branches were
lower than girdled branches 262 ng ml−1 versus 372 ng ml−1,
although this was not significant (P < 0.11; Figure 4). However,
unlike the stem girdling treatment, the drought treatment
induced a significant increase in xylem sap ABA concentrations
in the roots to 217 ng ml−1 (Figure 5C).
Stem girdling induced a significant reduction in NSC in root
tissues compared with the control after 35 days (Figure 7) with
root soluble sugars declining by 87% (8 versus 66 mg g−1 in
control and girdle treatments, respectively) and starch by 33%
(5 versus 8 mg g−1 in control and girdle treatments, respectively; Figure 7). In contrast to stem girdling, the drought treatment showed a slight reduction in root starch concentrations
compared with the control (P < 0.10), but no change in foliar
starch or foliar and root soluble sugar concentrations (Figure 7).
Discussion
ABA accumulation after girdling is linked to gas exchange
The cessation of phloem export by girdling across a single
branch as well as the entire canopy in P. radiata had significant
impacts on foliar ABA levels and gas exchange. In all three girdling experiments, an increase in foliar ABA level occurred within
days of girdling and was accompanied by reductions of gs in the
absence of a decline in foliar water status. The observed
increase in foliar ABA levels is consistent with a pattern of accumulation of foliar-synthesized ABA occurring under well-watered
conditions. Furthermore, a more rapid accumulation occurred
when the site of phloem obstruction was closer to the sites
where ABA could act on the guard cells, i.e. branch girdle. The
ABA-induced reductions in gs caused a decline in transpiration
and concomitant increase in leaf water potential at midday.
These patterns provide compelling evidence that the rates of
phloem export impact gas exchange via changes in foliar ABA
level. Our study suggests that a reduction in phloem export is
accompanied by a build-up of ABA in source tissues that is
largely responsible for a reduction in stomatal aperture in the
absence of a water potential trigger for ABA synthesis (Pierce
and Raschke 1980). This mechanism likely explains the commonly observed response of declining gs in trees subject to girdling (Myers et al. 1999, Iglesias et al. 2002, López et al. 2015).
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Figure 2. Girdling of single lateral branches affects foliar ABA levels in P. radiata. Response of stomatal conductance to fluctuating ABA levels in leaves
from control (open circles) and girdled branches (solid circles) in plants growing in the glasshouse (A) and under ambient, field conditions (D). Data
are fitted with a power function of the form y = a × xb (P < 0.05). Changes in foliar ABA level (±1 SE) through time (Day 0 represents start of treatment)
in girdled (solid bar) and control (open bar) at different sampling times in plants grown under glasshouse (B) and field conditions (E). Midday leaf water
potential (±1SE) for plants growing in the glasshouse (C) and under ambient, field conditions (F). Asterisks denote significant difference between treatments (P < 0.05).
6 Mitchell et al.
Figure 3. The response of (A) stomatal conductance, (B) light-saturated
photosynthesis and (C) internal [CO2] to stem girdling plants experiencing no change in water status (Day 21 of treatment). Asterisks denote
significant difference between treatments (P < 0.05).
Low canopy water status triggers ABA synthesis in foliage
While the site of synthesis for these fluctuations in foliar ABA
levels cannot be ascertained directly from these data, evidence
Table 1. Mean foliar NSC concentrations (±1 SE) for the three girdling experiments. Concentrations in bold denote significant differences between girdle and control samples (P < 0.05). Correlation coefficients for the soluble sugars versus stomatal conductance and starch versus stomatal conductance
for all sampling dates are also given.
Days since girdle Experiment
–2
22
35
9
50
7
58
Stem girdle (glasshouse)
Treatment Soluble sugars (mg g−1) Starch (mg g−1) Soluble sugars versus gs Starch versus gs
Control
Girdle
Control
Girdle
Control
Girdle
Branch girdle (glasshouse)
Control
Girdle
Control
Girdle
Branch girdle (field conditions) Control
Girdle
Control
Girdle
Tree Physiology Volume 00, 2016
56.9 (±2.1)
56.7 (±5.7)
60.1 (±5.7)
80.1 (±3.9)
57.2 (±1.5)
43.5 (±4.6)
71.6 (±5.4)
80.4 (±6.9)
52.8 (±2.9)
65.3 (±3.0)
83.2 (±4.9)
89.0 (±4.4)
80.0 (±3.0)
82.8 (±7.1)
7.5 (±0.4)
7.6 (±0.8)
8.3 (±0.6)
15.4 (±3.8)
7.2 (±0.3)
6.6 (±0.6)
9.13 (±1.4)
39.3 (±7.8)
9.9 (±1.8)
7.3 (±1.0)
14.9 (±2.6)
87.1 (±9.5)
15.5 (±1.7)
17.3 (±1.8)
R2 = 0.18
R2 = 0.01
R2 = 0.54
R2 = 0.57
R2 = 0.08
R2 = 0.01
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While a cessation in phloem export from the canopy induced an
increase in foliage/stem NSC, particularly starch, changes in NSC
did not consistently track changes in stomatal closure under the
different girdling treatments applied in this study (Table 1). The
observed increase in NSC tended to be transient and these
reserves were probably consumed for growth and/or respiration
under conditions of reduced source activity. Transient changes in
foliar NSC including starch concentrations may have reduced
photosynthetic capacity via several interrelated pathways after
girdling (Paul and Pellny 2003), yet our data demonstrate that
reductions in gs and their impacts on Asat were a direct result of
perturbations in foliar ABA level. Furthermore, declines in Asat were
accompanied by reductions in internal [CO2] in all but one of the
girdling treatments (where Asat remained similar to control levels),
suggesting that a large proportion of the changes in photosynthesis were due to stomatal limitation not photosynthetic performance. Nonetheless, we cannot exclude the possibility that the initial
carbohydrate accumulation may have induced some additional
ABA synthesis via mechanisms associated with sugar-sensing
enzymes such as hexokinase (León and Sheen 2003, Wingler and
Roitsch 2008, Kelly et al. 2013) or the production of reactive oxidative species (Galvez-Valdivieso et al. 2009). Elevated ABA
levels in foliage have also been shown to reduce leaf hydraulic
conductance (Kleaf) upstream of stomata thereby reducing stomatal conductance indirectly via a ‘hydraulic’ effect (Pantin et al.
2013). While this mechanism cannot be ruled out, recent studies
tracking patterns of leaf ABA, gs and Kleaf have indicated no appreciable declines in Kleaf under increasing ABA during fluctuations in
atmospheric demand (McAdam and Brodribb 2015).
Contribution of foliar-derived ABA to gas exchange
from the root and foliar comparisons at the end of the stem girdling experiment suggest predominance of foliage-derived ABA in
controlling canopy gas exchange when plant water potential
declines. The longer term (21–35 days) consequences of stem
girdling were a significant depletion of root NSC and a corresponding decline in predawn and midday Ψl and stem water content providing clear evidence that root water uptake was
compromised and canopy water stress developed without a
change in soil water status. The low NSC concentrations in roots
likely reduced the metabolic energy required for root water uptake
and ABA biosynthesis as demonstrated by Manzi et al. (2015).
Under these conditions, foliar rather than root ABA levels
increased in response to low foliar water potential. It is unlikely
that an undetected source of ABA conjugates (e.g. ABA-GE)
synthesized in the roots was responsible for the very large spike in
foliar free ABA, as increases in both forms tend to occur together
despite their relative contributions differing among species and
treatments (Sauter et al. 2002). The consistent increase in foliar
and shoot ABA level, independent of root xylem concentrations,
confirms our hypothesis that production of ABA in the canopy was
independent of root ABA levels and sufficient to induce stomatal
Figure 5. Response of xylem sap ABA concentrations (±1 SE) under different experimental treatments inducing different changes in leaf water
potential. (A) Leaf water potential (predawn) when xylem sap ABA concentrations were sampled (±1SE, n = 3), (B) sapwood water content
(±1 SE, n = −6) and (C) xylem sap ABA concentration expressed in
roots and shoots for stem girdle (at 41 days) and drought treatments (at
33 days) and control (±1 SE, n = 3). Note girdled shoots were also
sampled in the drought experiment. Asterisks denote significant differences between treatments.
closure. This provides strong evidence that foliage-derived ABA is
critical for stomatal regulation in P. radiata and is in agreement
with several other studies involving herbaceous and woody angiosperm species (Soar et al. 2006, Christmann et al. 2007, Ikegami
et al. 2009, McAdam et al. 2016b).
Under conditions of soil water deficit sufficient to induce stomatal closure, root and canopy ABA levels tended to increase in
concert in ungirdled plants. This result indicates that ABA synthesis occurs across different organs simultaneously and/or the
long-distance transport of ABA across the plant can augment
ABA levels in organs that do not actively synthesize ABA in
response to water deficit (Manzi et al. 2015). For example,
exchange of ABA from the xylem to the phloem may also
enhance root ABA concentrations under water stress given that
whole-plant studies of ABA fluxes showed that a significant pool
of shoot ABA can be recirculated basipetally via the phloem
(Wolf et al. 1990, Manzi et al. 2015). Regardless of the source
of increase in root ABA levels under water deficit, our girdling
results demonstrate a significant source of locally synthesized
(foliar) ABA for the control of gas exchange, a mechanism that
most likely causes stomatal closure when water potential
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Figure 4. Drought triggers an accumulation of foliar ABA level and a
reduction in stomatal conductance. (A) Response of stomatal conductance to rising foliar ABA levels in droughted plants with branch-girdled
(solid triangles) and non-girdled (solid circles) branches and control
plants (open circles). Data are fitted with a power function of the form
y = a × xb. (B) Changes in foliar ABA level (±1 SE) after the imposition
of drought (Day 0) in different treatments. Leaf water potential (±1SE)
for (C) predawn and (D) midday measurements in different treatments.
Lower case letters denote significant difference between treatments and
sampling times (P < 0.05).
7
8 Mitchell et al.
Figure 7. Changes in NSC concentrations in response to stem girdling
and drought. (A) Soluble sugar and (B) starch concentration (dry weight
basis) for root and foliage in P. radiata after 38 days of stem girdling and
34 days of withholding water.
declines are also driven by soil water deficit. The trigger for ABA
production in this study was closely related to canopy water status when stem girdling or soil water deficit reduced water uptake
(Figure 6), suggesting strong coordination between foliar ABA
levels and Ψl across a range of soil water contents.
VPD and transpiration. These rapid declines in foliar water status
often induce short-term changes in foliar ABA concentrations that
provide the direct and localized trigger for a reduction in transpiration (Correia et al. 1997, Thomas and Eamus 2002).
Furthermore, if the pool of ABA synthesized in foliage represents
the major source of whole-plant ABA, then those factors affecting
foliar production and phloem export of ABA for the plant become
important for many physiological functions beyond stomatal control. For example, changes in ABA concentrations in phloem sap
may also affect rates of phloem unloading in sink tissues (Hoad
1995), thereby influencing root growth and development
(McAdam et al. 2016a).
The significance of foliar-derived ABA for tree water and
carbon relations
This study suggests that changes in both phloem export and leaf
water status that are independent of root water status can provide
different and sometimes interrelated mechanisms for regulating
stomata via increases in ABA levels in the foliage. The strong role
of foliar-derived ABA in regulating gas exchange in P. radiata suggests that root–shoot signalling is not always a critical component
of the regulation of stomatal responses to water stress. If this is
applicable to tree species more broadly, it signifies a greater role
of ABA signalling for regulating carbon and water exchange in
tree species than what is currently acknowledged. Root-mediated
signalling of ABA is reliant on root water status declining before
changes in water status in the canopy. Because hydraulic signals
across the soil–plant–atmosphere continuum can be propagated
almost instantaneously, tree canopies may experience significant
diurnal declines in water potential in response to fluctuations in
Tree Physiology Volume 00, 2016
Acknowledgments
We would like to thank Rachael Berry for assistance with ABA
extraction and Renee Smith and Kaushal Tewari for performing
the carbohydrate analyses.
Conflict of interest
None declared.
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Figure 6. Foliar ABA level is a function of leaf water potential in P. radiata
exposed to drought. The response of foliar ABA levels to (A) predawn
and (B) midday leaf water potential in needles from droughted (girdled
and non-girdled branches) and control plants.
Contribution of foliar-derived ABA to gas exchange
Funding
Funding for this work was from the National Centre for Future
Farm Industries (under a secondment to University of Tasmania
for P.J.M.) and the Australian Research Council (DE140100946,
postdoctoral fellowship to S.A.M.M.).
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Tree Physiology Volume 00, 2016