Journal of Experimental Botany, Vol. 68, No. 9 pp.

2413–2424, 2017
doi:10.1093/jxb/erx116 Advance Access publication 17 April 2017
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Applying ‘drought’ to potted plants by maintaining

suboptimal soil moisture improves plant water relations

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
Jaime Puértolas*, Elisabeth K. Larsen†, William J. Davies and Ian C. Dodd
The Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK

* Correspondence: j.puertolas@lancaster.ac.uk
†
Present address: Fundación de la Comunitat Valenciana Centro de Estudios Ambientales del Mediterráneo, Parque Tecnológico, C/
Charles Darwin 15, 46980, Paterna, Valencia, Spain.

Received 24 January 2017; Editorial decision 17 March 2017; Accepted 21 March 2017

Editor: Christine Raines, University of Essex

Abstract
Pot-based phenotyping of drought response sometimes maintains suboptimal soil water content by applying high-
frequency deficit irrigation (HFDI). We examined the effect of this treatment on water and abscisic acid (ABA) relations
of two species (Helianthus annuus and Populus nigra). Suboptimal soil water content was maintained by frequent
irrigation, and compared with the effects of withholding water and with adequate irrigation. At the same average
whole-pot soil moisture, frequent irrigation resulted in larger soil water content gradients, lower root and xylem ABA
concentrations ([X-ABA]), along with higher transpiration rates or stomatal conductance, compared with plants from
which water was withheld. [X-ABA] was not uniquely related to transpiration rate or stomatal conductance, as fre-
quently irrigated plants showed partial stomatal closure compared with well-watered controls, without differing in
[X-ABA] and, in H. annuus, [ABA]leaf. In two P. nigra genotypes differing in leaf area, the ratio between leaf area and
root weight in the upper soil layer influenced the soil water content of this layer. Maintaining suboptimal soil water
content alters water relations, which might become dependent on root distribution and leaf area, which influences soil
water content gradients. Thus genotypic variation in ‘drought tolerance’ derived from phenotyping platforms must be
carefully interpreted.

Key words: ABA, drought, frequent irrigation, genotype screening, Helianthus annuus, phenotyping platform, Populus nigra, soil
moisture heterogeneity.

Introduction
The study of plant responses to drought is becoming even the main research targets to increase crop yield under lim-
more relevant under the current uncertainties regarding food ited water availability (Chaves and Davies, 2010), but plant
and energy security under a changing climatic scenario. There responses to water deficit are complex and the traits control-
is a growing pressure on regional water resources to maintain ling those responses can have different impacts depending on
food and biofuel crop yield and an urgent need to increase the drought scenario (Tardieu, 2012). Thus assessing geno-
agricultural production on marginal lands prone to drought type performance under drought is problematic, especially
conditions. Selecting drought-tolerant genotypes is one of when most studies simply withdraw water and quantify plant

Abbreviations: ABA, abscisic acid; [ABA]leaf, abscisic acid concentration in leaf tissue; [ABA]root, abscisic acid concentration in root tissue; D/RW, dry and rewetting
irrigation treatment; gs, stomatal conductance; HFDI, high-frequency deficit irrigation; PWsat, pot weight at soil saturation; [X-ABA]root, abscisic acid concentration
in root xylem sap; [X-ABA]shoot, abscisic acid concentration in shoot xylem sap; WW, well-watered irrigation treatment; ψbulkroot, bulk root water potential; ψroot, local
root water potential; ψpd, pre-dawn leaf water potential; ψshoot, shoot water potential.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which per-
mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
2414 | Puértolas et al.

survival (Lawlor, 2013; Blum, 2014). Nowadays, powerful heterogeneity by manipulating either irrigation placement
statistical tools can associate genes with specific phenologi- (partial rootzone; Dodd et al., 2008a) or timing (irrigation fre-
cal traits, including physiological responses to water defi- quency experiments; Boyle et al., 2016b) can diminish xylem
cit (Baginsky et al., 2010; Hu and Xiong, 2014), which can ABA concentration, although other reports show that ABA
improve the breeding process or even be used within genetic levels might be regulated by overall soil water content (Einhorn
modification programmes. Technologically advanced high- et al., 2012; Puértolas et al., 2013). The impact of the irrigation
throughput screening platforms equipped with automated procedure in phenotyping platforms needs to be assessed to
irrigation systems can measure a wide range of morpho-phys- understand the physiological impacts of different ABA levels.
iological variables in a large number of genotypes to obtain This study aimed to determine the effect of frequent irri-
the necessary data for those analyses (Furbank and Tester, gation applied to maintain constant suboptimal soil water

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
2011; Neilson et al., 2015). However, the effects of experimen- content in drought experiments on plant water and ABA rela-
tal manipulations in controlled conditions on plant physiol- tions. The objective was to assess the impact of this proce-
ogy, in particular those performed in potted plants, need to be dure on phenotype screening for drought resistance traits. For
carefully examined to determine whether similar physiologi- that purpose, two experiments in two different species com-
cal responses occur in field-grown plants (Poorter et al., 2012, pared plant physiological responses to three irrigation pro-
2016). There are different approaches to restrict water when cedures: daily replacement of transpirational losses to ensure
screening for drought tolerance (Passioura, 2012). The use of well-watered plants; periodic drying and complete rewetting
automated systems favours the use of frequent or continuous cycles; and daily irrigation to maintain suboptimal soil mois-
weighing of pots, with frequent irrigation aiming to restore ture. Plants in the two deficit treatments were measured at
a pre-determined pot weight corresponding to a specific soil the same whole-pot soil water content. Two contrasting spe-
moisture target, over sustained periods of time (Granier cies were chosen to assess the consistency of the observed
et al., 2006; Pereyra-Irujo et al., 2012; Tisne et al., 2013). This responses. Helianthus annuus is a herbaceous species propa-
method was discussed and defined by Blum (2011) as being gated from seeds described in previous reports as typically
of questionable physiological relevance in phenotyping geno- anisohydric, while Populus nigra is a woody isohydric species
typic variation in drought resistance. propagated from cuttings (Tardieu and Simonneau, 1998).
Irrigation frequency can determine plant water relations The first experiment explored the effect of these treatments
and soil water availability. Under optimal conditions, high- on H. annuus at different times of the day, and the second
frequency irrigation enhanced bulk soil water content, and assessed these effects on two different genotypes of P. nigra
therefore root water uptake efficiency and yield of lysimeter- with contrasting leaf area to test whether genotypic differ-
grown sunflower (Helianthus annuus) plants (Segal et al., ences in plant water uptake influence the observed responses.
2006). Moreover, irrigation frequency at suboptimal soil Two questions were addressed. (i) For equivalent subopti-
moisture can greatly influence water relations of potted mal soil moisture, how does high-frequency deficit irrigation
plants. Daily watering of potted Pelargonium hortorum plants (HFDI) modify water and ABA relations compared with
with 50% of the water applied to well-watered control plants plants subjected to drying cycles? (ii) Do genotypic differ-
decreased abscisic acid (ABA) concentration in the xylem ences in water use explain plant responses to HFDI?
sap and increased transpiration rates and leaf water poten-
tial compared with plants receiving the same irrigation vol-
ume but applied cumulatively every 4 d (Boyle et al., 2016a). Materials and methods
Understanding the effects of different irrigation regimes
within phenotypic screening is needed, as the method of Two experiments were performed. Experiment 1 assessed the effect
of maintaining suboptimal soil moisture by applying HFDI on daily
imposing soil water deficit might modify genotype rankings variation in ABA levels in H. annuus. Experiment 2 studied the effect
of drought tolerance traits. In these experiments, since the vol- of maintaining suboptimal soil moisture on ABA signalling in two
ume of water added in each irrigation is proportional to the genotypes of P. nigra with contrasting leaf area.
water lost since the previous irrigation, differences in water
uptake between genotypes can determine soil moisture distri- Plant material, growing conditions, and irrigation treatments
bution and thus genotypic differences in drought responses. In Experiment 1, two sunflower (H. annuus) seeds were sown in each
For example, genotypes with a larger leaf area, usually with of 64 square section pots (6×6×30 cm high; 1.1 litre volume), with a
higher water uptake, would need to receive more water in perforated (9 mm diameter holes) plastic sheet glued to one end to
each irrigation event, which could change water distribution hold soil while allowing drainage. After germination, a single plant
in the pot compared with genotypes receiving less water. was left in each pot.
In Experiment 2, P. nigra hardwood cuttings from each of two
One of the physiological traits that may discriminate spe- genotypes were planted in wet perlite after dipping the basal end in a
cies differences in water use strategies is ABA production in 1 mM indole-3-butyric acid solution to facilitate rooting. Genotypes
response to soil drying (Sreenivasulu et al., 2012). However, were selected based on contrasting leaf morphology and total leaf
the effects of ABA on different aspects of plant physiology are area. Genotype B had larger leaves and higher total leaf area than
complex and often contradictory (Saradadevi et al., 2014), and genotype S. After 1 month, 32 rooted cuttings from each genotype
were transplanted to cylindrical pots (6.5 cm in diameter, 21 cm in
finding an ABA ideotype (Blum, 2015) requires an understand- height, 0.8 litre volume) with a stainless steel mesh (0.7 mm aper-
ing of how this hormone is produced and transported within ture) at one end to assist drainage. The pot was designed to fit in the
the plant. Experiments inducing pronounced soil moisture pressure chamber of the same volume.
 Drought experiments in potted plants and water relations | 2415

In both experiments, pots were cut lengthwise in two halves and

stuck together with duct tape to facilitate root and soil extraction
at harvest. Pots were filled with an organic loam (John Innes No2,
J. Arthur Bowers, UK) up to 3 cm from the top of the pot.
Plants were grown in a walk-in controlled environment cham-
ber, under the following environmental conditions: photosyntheti-
cally active radiation (PAR)=400 µmol m–2 s–1 provided by halogen
lamps (HQI-BT 400W/D, Osram, Germany); day/night tempera-
ture=24/16 °C; and photoperiod=16 h in Experiment 1, and 14 h in
Experiment 2 to prevent excessive daily transpiration. At the begin-
ning of the experiment, pots were watered to field capacity at the end
of the photoperiod, left to drain overnight, and weighed to obtain

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
weight at pot capacity (PWsat). Pots were weighed twice a day during
the whole experiment and watered according to pot weight. Plant
weight was neglected as it was <5% of the water weight at the lower
soil moisture threshold.
Plants were watered to PWsat daily for the first 3 weeks in
Experiment 1, and 2 weeks in Experiment 2. After that period, they
were randomly assigned to the following three irrigation treatments
(Fig.1).

WW
Plants were watered daily during the first 3 d and then twice a day to
PWsat (16 plants in Experiment 1; 12 plants in Experiment 2, six per
genotype). Watering was carried out in the morning and after mid-
day (2–3 h and 8–9 h after the start of the photoperiod, respectively).

D/RW
For the drying and rewetting cycle (24 plants in Experiment 1; 28 in
Experiment 2, 14 per genotype), water was withheld until pot weight
reached a threshold (a soil moisture that halved stomatal conduct-
ance (gs) compared with WW plants) and then re-watered to reach
PWsat.. In Experiment 1, this threshold was set at PWsat–275 g, which
corresponded to an average soil water content of 0.16 g g–1 and a soil
matric potential of –0.13 MPa according to a soil moisture release
curve previously performed on the same substrate (Puértolas et al.,
2013). In Experiment 2, this threshold was PWsat–160 g (0.10 g g–1,
corresponding to a soil matric potential of –1.09 MPa). Pots were
weighed with the same frequency as WW plants, but watered only if
weight was below the minimum threshold set. Plants were subjected
to 2–3 cycles during the experiment.

Fig. 2. Whole-pot gravimetric soil water content (θg)(A), stomatal

conductance (gs) (B), shoot water potential (ψshoot) (C), and ABA
Fig. 1. Evolution of whole-pot soil gravimetric water content (θg) during concentration in shoot xylem sap ([X-ABA]shoot) (D) in Helianthus annuus in
Experiment 1 in one example plant of Helianthus annuus per irrigation different irrigation treatments (HFDI, patterned; D/RW, black; WW, white) at
treatment measured for 10 d after the start of the treatments (WW, different times of the day. Data are means ±SE of eight replicates for HFDI
continuous line; HFDI, dashed line; D/RW, dotted line). Total water applied and D/RW, and six for WW. P-values from the ANOVA are shown for each
for each treatment is shown. Measurements were taken at the end of the variable. Different letters denote significant differences between irrigation
treatment application in all plants. treatments within each time of the day (Tukey, P<0.05)
2416 | Puértolas et al.

HFDI genotype (data not shown), we decided not to measure it during the
Water was withheld as in D/RW, and then plants were re-watered experiment and report only the whole-plant transpiration rate as a
twice a day (as necessary) to reach the minimum threshold set in measure of stomatal control of water losses.
each experiment (24 plants in Experiment 1; 28 in Experiment 2, 14
per genotype).
Plant and soil water relations
One leaf from the upper third of the canopy (in Experiment 1, one
Measurement design of those where gs was measured for morning and midday measure-
In Experiment 1, measurements were made over 4 d, starting 4 ments) was excised, immediately frozen in liquid nitrogen, and stored
weeks after germination. On each day, two plants of D/RW and at –20 °C. Then the plant was de-topped, and the shoot placed in a
HFDI treatments (eight in total during the 4 d) and 1–2 plants of pressure chamber (Soil Moisture Equipment Corp., Santa Barbara,
WW (5–6 plants in total) were measured immediately before the CA, USA) to measure shoot water potential (ψshoot). In Experiment

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
start of the photoperiod (pre-dawn), 1 h after the start of the photo- 2, pre-dawn leaf water potential was also measured in all harvested
period, immediately before the morning irrigation, and 7 h after the plants immediately before the start of the photoperiod (ψpd) and
start of the photoperiod, immediately before the midday irrigation. pot weight was recorded simultaneously to estimate soil water con-
WW and HFDI plants were randomly selected, while D/RW plants tent. After reaching the balancing pressure, an overpressure of up
were selected according to pot weight to ensure they were below the to 0.5 MPa was applied to allow sap collection. Sap was immedi-
PWsat–275 g threshold at the time of measurement, in order that ately frozen in liquid nitrogen and stored at –20 °C for subsequent
whole-pot soil moisture was comparable with HFDI plants. determination of the ABA concentration in the shoot xylem sap
In Experiment 2, plants were measured over 6 d, starting 4 weeks ([X-ABA]shoot). Each pot was opened and roots were extracted from
after transplanting. For each measurement day, one plant per geno- each of the three soil column layers (10 cm and 7 cm in length for
type of WW and 2–3 plants of D/RW and HFDI treatments were Experiments 1 and 2, respectively). Roots were quickly washed
selected for measurements between 2 h and 7 h after the start of the (<60 s), blotted, frozen in liquid nitrogen, and stored at –20 °C.
photoperiod. A soil sample of each layer was weighed, oven-dried at 70 °C until
constant weight was reached, and weighed again to calculate soil
gravimetric water content in each section (θg).
Plant water use Additionally, in Experiment 2, after shoot removal to measure
In Experiment 1, gs was measured in the two most apical fully ψshoot, half of the pots (three WW and seven D/RW or HFDI for
expanded leaves with a porometer (AP4, Delta-T, Burwell, UK) and each genotype) were inserted in the pressure chamber to determine
averaged. Measurements were not taken at pre-dawn. bulk root water potential (ψbulkroot) and extract root xylem sap. After
In Experiment 2, for each plant, both ends of the pot were covered measuring ψbulkroot, pressure was increased at 0.04 MPa intervals. At
with duct tape, and pot weight was recorded 1 h before measure- each step, sap was collected for 20 s in a pre-weighed Eppendorf
ment. It was weighed again 1 h later to calculate the plant water tube and weighed to calculate the sap flow rate. This sap flow rate
uptake rate. Total leaf area was measured in both experiments was compared with the actual whole-plant transpiration rate and
with a leaf area meter (Li-3100C, Licor, Lincoln, NE, USA) and, pressure was increased until the two values matched. Sufficient
in Experiment 2, plant transpiration rate was calculated as water sap (>50 µl) was collected at the matching flow rate to determine
uptake rate divided by leaf area. Preliminary gs measurements were root xylem ABA concentration ([X-ABA]root). The remaining pots,
made at the beginning of the experiment. Since we found high vari- which were not pressurized, were opened and a root sample (2 cm in
ability across the canopy of each single plant, especially in the S length) was excised from each of three layers within the soil column

Table 1. Root dry weight (mean ±SE; mg) in each column layer for the three irrigation treatments and two species

In parentheses, the percentage of the root dry weight in the layer with respect to total dry weight is given.

Upper layer Middle layer Lower layer Total

H. annuus WW 40.5 ± 4.9 a 30.8 ± 4.1 a 33.7 ± 4.0 a 105.0 ± 12.0 A
(40.0 ± 2.3 b) (28.6 ± 1.8 a) (31.4 ± 2.1 a)
HFDI 29.9 ± 4.2 b 21.8 ± 3.5 a,b 18.0 ± 3.4 a 69.7 ± 7.5 A
(42.4 ± 2.9 b) (32.1 ± 2.1 a) (25.5 ± 2.1 b)
D/RW 32.0 ± 4.2 a 27.6 ± 3.5 a 26.1 ± 3.4 a 85.7 ± 10.3 A
(39.2 ± 2.6 b) (31.0 ± 1.6 a) (29.7 ± 2.5 a)
Average 33.5 ± 2.6 b 26.3 ± 2.1 a,b 25.1 ± 2.2 a
(40.6 ± 1.3 b) (30.8 ± 1.1 a) (28.6 ± 1.5 a)
P. nigra WW 239 ± 30 a,b 178 ± 18 a 334 ± 46 b 750 ± 70 A
(31.8 ± 2.7 a) (23.9 ± 1.5 a) (44.4 ± 3.7 b)
HFDI 264 ± 35 b 158 ± 12 a 338 ± 22 c 760 ± 39 A
(35.7 ± 2.2 b) (20.4 ± 0.9 a) (43.8 ± 2.1 c)
D/RW 294 ± 28 b 206 ± 18 a 406 ± 28 c 904 ± 60 B
(31.9 ± 1.7 b) (22.4 ± 1.2 a) (45.7 ± 1.4 c)
Average 271 ± 15 b 181 ± 10 a 365 ± 17 c
(33.4 ± 1.3) (21.9 ± 0.7 a) (45.7 ± 1.2 c)

For each species, different lower case letters denote statistical differences between layers within each irrigation treatment and within the average
across treatments. Upper case letters denote differences between irrigation treatments for total weight (Tukey, P<0.05)
 Drought experiments in potted plants and water relations | 2417

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
Fig. 3. Soil gravimetric water content (θg) (A) and ABA concentration in roots ([ABA]root) (B) in Helianthus annuus in different irrigation treatments and soil
layers (0–10 cm, white bars; 10–20 cm, patterned bars; 20–30 cm, black bars). Data are means ±SE of eight replicates for HFDI and D/RW, and six for
WW. P-values for irrigation, soil layer, and their interaction in the repeated measures ANOVA are shown for each variable. Time and its interactions were
not significant for either variable. Different lower case letters denote significant differences between depth×irrigation treatment combinations, while upper
case letters denote differences of the average across depths between irrigation treatments (Tukey, P < 0.05).

Table 2. Results of the ANOVA for variables measured in

Experiment 2 which are not shown in the figures

Variable Source of variation df F P

Leaf area Irrigation 2 3.0 0.06
Genotype 1 32.9 <0.001
I×G 2 0.9 0.40
ψpredawn Irrigation 2 10.7 <0.001
Genotype 1 5.1 0.03
I×G 2 1.7 0.14
[ABA]leaf Irrigation 2 3.6 0.03
Genotype 1 0.5 0.49
I×G 2 0.8 0.46

ψpredawn, pre-dawn water potential; [ABA]leaf, ABA concentration in

leaf tissue.
In bold, statistical significance (P<0.05).

by determining the voltage output versus water potential relation-

ship using four different NaCl concentrations of known osmotic
potential.

ABA determination
Stored leaf and root tissues were freeze-dried and finely ground to
determine [ABA]leaf and [ABA]root. Root dry weight in each layer
was determined before grinding. The ground tissue was incubated
in distilled water (1:50, w/w) at 4 °C overnight in a shaker. ABA
concentrations in xylem sap and aqueous extracts of tissues were
analysed by a radioimmunoassay (Quarrie et al., 1988). Based on
a cross-reactivity test (Quarrie et al., 1988), there was no non-spe-
Fig. 4. Relationship between local soil gravimetric water content (θg) and cific interference during the assay for either leaf or root extracts of
ABA concentration in the roots ([ABA]root) for each irrigation treatment P. nigra and H. annuus.
(HFDI, grey circles; D/RW, black circles; WW, white triangles) in Helianthus
annuus (A) and Populus nigra (B). Each point represents paired samples
taken within a layer within a plant. Statistical analyses
For both experiments, variables not associated with position in the
(7 cm in length), tapped to remove adhering soil particles, blotted, soil column were analysed by two-way ANOVA, with the factors
and quickly inserted (<15 s between excision and insertion) in a psy- comprising irrigation treatment (both experiments), time of the day
chrometric chamber (C-52, Wescor, Logan, UT, USA) to equilibrate for Experiment 1 and genotype for Experiment 2, with day of meas-
before measuring ψroot. Measurements were made by dew-point psy- urement as a block. Variables associated with vertical position in the
chrometry. Each psychrometric chamber was previously calibrated soil column ([ABA]root, root dry weight, θg, ψroot) were analysed by
2418 | Puértolas et al.

repeated measures ANOVA, with soil layer as the repetition factor.

Additionally, the possible effect and interactions of pressurization on
[ABA]root in Experiment 2 were analysed by introducing that factor
in the analysis. Since neither pressurization nor the interactions with
treatment or soil layer were significant, data for both pressurized
and non-pressurized pots were included in the analysis. When inter-
actions were significant, separate one-way ANOVAs were applied
comparing irrigation treatments in each of the levels of the other
factor. To account for variability in plant leaf area in Experiment
2, one-way analysis of covariance (ANCOVA) was performed, with
leaf area as covariate and irrigation as a factor using only the two
water deficit treatments. One-way ANCOVA was also used to com-

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
pare the slope of the relationship of ψroot and [X-ABA]root with tran-
spiration rate between two data sets: WW+HFDI and WW+D/RW.
In all cases, a post-hoc test discriminated differences between irriga-
tion treatments (Tukey HSD, P<0.05).

Results
Experiment 1. Helianthus annuus
At harvest, leaf area was higher (P<0.001) in WW (572 ± 32
cm2) than in D/RW (353 ± 26 cm2) and HFDI (343 ± 26 cm2)
plants, with no significant differences between the two latter
treatments.
Whole-pot soil water content was 50% lower in D/RW and
HFDI compared with WW, but values were similar in D/RW
and HFDI at any time of the day, as intended (Fig. 2A). gs was
higher at midday than in the morning (Fig. 2B). WW plants
had higher gs than the other treatments, but differences were
larger at midday. Morning measurements revealed no statisti-
cal differences in gs between HFDI and D/RW, but at mid-
day HFDI had an 80% higher gs than D/RW. Irrigation, time
of the day, and their interaction significantly affected ψshoot,
which was always higher for WW plants independent of the
time of measurement (Fig. 2C). Pre-dawn ψshoot was 0.15 MPa
lower for D/RW than HFDI plants, but there were no signifi-
cant differences in ψshoot in the morning and at midday. Foliar
ABA concentration was significantly higher (P=0.001) in D/
RW plants (6.2 ± 0.9 nmol g–1 DW) than in WW and HFDI
plants, which showed similar values (3.0 ± 0.5 nmol g–1 DW
and 2.9 ± 0.4 nmol g–1 DW for WW and HFDI, respectively).
Leaf xylem ABA concentrations showed similar patterns
to foliar ABA accumulation (Fig. 2D). Although treatment
effects on gs and ψshoot varied according to the time of day
(Fig. 2B, C), xylem ABA concentration was always higher in
D/RW than in WW and HFDI plants.
Soil moisture varied between soil layers in all treatments,
with the highest soil water content in the upper 10 cm for
WW and HFDI plants, while in D/RW plants the basal 10 cm
had a slightly higher θg. Root dry weight was higher in the
upper 10 cm (Table 1). Averaging across soil layers, [ABA]root
Fig. 5. Shoot water potential (ψshoot) (A), bulk root water potential (ψbulkroot)
decreased in the order: D/RW>HFDI>WW (Fig. 3B). (B), transpiration rate (Tr) (C), and ABA concentration in shoot xylem sap
However, a strong irrigation×layer interaction was observed. ([X-ABA]shoot) (D) in Populus nigra in different irrigation treatments and
In WW and HFDI, [ABA]root was lower in the upper 10 cm genotypes (genotype B, black bars; genotype S, white bars). Data are
than in the other two lower layers, while in D/RW there were means ±SE of 14 replicates for HFDI and D/RW, and 6 for WW, except
no significant differences across soil layers. For a similar level for ψroot, where n=7 for HFDI and D/RW, and n=3 for WW. P-values from
the ANOVA are shown for each variable. Different letters denote significant
of local soil water content, [ABA]root in HFDI was lower than differences between irrigation treatments for the average of the two
in D/RW (Fig. 4A). No statistical differences between time of genotypes, as the genotype×irrigation interaction was not significant for
the day were found for θg, root dry weight, or [ABA]root. any variable (Tukey, P<0.05).
 Drought experiments in potted plants and water relations | 2419

Experiment 2. Populus nigra Leaf area did not affect transpiration rate. However, within
the two deficit irrigation treatments, water uptake was related
Leaf area was 36% higher in genotype B than in genotype S to the leaf area:root weight ratio, but the relationship differed
(Table 2) and tended to be smaller (12%) in HFDI than in D/ (P=0.01 for the leaf area×treatment interaction). While water
RW and WW plants. uptake was not correlated with the leaf area:root weight ratio
ψpd was higher in WW (–0.14 ± 0.03 MPa) than in HFDI in D/RW, it decreased in HFDI (Fig. 7A). Plant water uptake
(–0.42 ± 0.07 MPa) and lowest in D/RW (–0.67 ± 0.08 MPa) was also positively related to soil moisture in the upper soil
(Table 2), even though whole-pot soil water content at the layer (0–7 cm) for HFDI but not for D/RW (P=0.001 for the
time of measurement was higher in D/RW than in HFDI (but water uptake×treatment interaction) (Fig. 7D). Consequently,
HFDI had higher local soil water content in the upper layer). ψbulkroot (Fig. 7B) and soil moisture in the upper layer (Fig. 7C)
Averaged across treatments, ψpd was 0.25 MPa lower in geno-

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
decreased with the leaf area:root weight ratio in HFDI, while
type B than in genotype S, even though whole-pot soil water no relationship was observed in D/RW. For the two lower lay-
content was similar (P=0.67) between genotypes. ers, soil moisture was not related to either leaf area or water
Overall variability in both ψpd and ψbulkroot within HFDI uptake. Overall, these results suggest that the leaf area:upper
P. nigra plants was much higher than in ψpd for H. annuus, with root weight ratio determined soil moisture gradient, ψbulkroot,
a higher coefficient of variation (CV) for P. nigra (CV=0.93 and, in turn, transpiration rate.
for both ψpd and ψbulkroot in P. nigra; CV=0.11 for ψpd in Whole-pot soil water content was higher in WW and simi-
H. annuus). Genotype did not alter the responses of shoot lar in D/RW and HFDI (P<0.001). It was significantly higher
and bulk root water potential to the irrigation treatments in genotype S (P=0.02), with no genotype×irrigation interac-
(no significant genotype×treatment interaction). Although tion (P=0.83). Vertical soil moisture profiles differed between
genotype did not affect ψbulkroot, ψshoot was –0.25 MPa lower treatments (a highly significant soil layer×irrigation treat-
in genotype B (averaged across treatments). Both ψshoot and ment interaction for θg; P<0.001). Soil moisture was similar
ψbulkroot were lowest in the D/RW treatment, with no signifi- in all layers for D/RW plants, but strongly decreased from the
cant differences between HFDI and WW plants (Fig. 5A, B). upper wetter layer to the drier lower layer in HFDI plants
Transpiration rate (normalized per unit leaf area) was (Fig. 8A). Root dry weight distribution was similar across
affected by irrigation treatment, but not by genotype or irrigation treatments: lowest in the middle and highest in the
genotype×treatment interaction. Transpiration of HFDI and lower layer (Table 1). Genotype B had 46% more root bio-
D/RW plants was 30% and 60% of WW plants, respectively mass than genotype S (P<0.001), with a greater difference in
(Fig. 5C). Foliar ABA concentration ([ABA]leaf) was lower the lower layer (P=0.001 for depth×genotype interaction). D/
(P=0.03) in WW (6.2 ± 0.4 nmol g–1 DW) than in D/RW RW plants had 20% more root biomass than the other two
(8.3 ± 0.6 nmol g–1 DW) and HFDI (8.1 ± 0.4 nmol g–1 DW) treatments.
plants. ABA concentration in shoot xylem sap ([X-ABA]shoot) Root water potential did not differ between soil layers in
was higher only in D/RW, with no statistical differences WW and D/RW plants, and was on average 0.4 MPa higher
between HFDI and WW (Fig. 5D), even though average in WW plants. There was a pronounced ψroot gradient within
[X-ABA]shoot was 2- to 3-fold higher in HFDI, due to great the soil column in the HFDI treatment (Fig. 8B), with the
variability within this treatment. Transpiration rate decreased upper layer having a similar ψroot to WW plants. Root ABA
with both ψbulkroot and [X-ABA]root. However, the slope of the concentration was highest in D/RW and lowest in WW
decrease with respect to WW was steeper in HFDI than in D/ plants (Fig. 8C), with no genotype or genotype×irrigation
RW (Fig. 6). interaction. In HFDI, it was 30% higher in the lower layer

Fig. 6. Relationship between transpiration rate and (A) bulk root water potential (ψbulkroot) and (B) ABA concentration in the shoot xylem sap ([X-ABA]shoot)
for each irrigation treatment (HFDI, black triangles; D/RW, black circles; WW, white triangles) in Populus nigra. The regression line for WW and HFDI
(dashed) and WW and D/RW (dotted) pools is shown in both panels. P-values and the results of the ANCOVA to compare regression slopes are shown.
2420 | Puértolas et al.

than in the other two layers, which had similar values to

WW plants. In D/RW and WW plants, [ABA]root was lower
in the lower layer of the column. In contrast to sunflower,
[ABA]root increased in the driest layers similarly in HFDI
and D/RW.

Discussion
HFDI increased ψroot and decreased [X-ABA]shoot in com-

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
parison with plants from which water was withheld, at the
same whole-pot soil moisture content (Fig. 9). Thus this irri-
gation procedure might not be appropriate when screening
for stomatal responses to drought, as it improves root water
status and suppresses xylem ABA concentration. Therefore,
the results from screening using HFDI must be taken with
caution. For example, the low [X-ABA]shoot in sunflower was
not related to decreased gs in comparison with well-watered
plants (cf. Fig. 2B, D), in contrast to previous predictions for
sunflower (Tardieu and Simonneau, 1998) where [X-ABA]shoot
is uniquely related to gs. Even though the radioimmunoassay
method used to quantify ABA concentration might not as
readily detect subtle differences unlike more recent meth-
ods based on HPLC (McAdam, 2015), the clear differences
between drought treatments found in this study demonstrate
this differential effect of HFDI on the ABA versus stoma-
tal conductance relationship. Moreover, since ψbulkroot or ψpd
determine gs (Fig. 6A) and other physiological traits (such as
[ABA]root), different effects of HFDI in different genotypes
can impact on phenotype screening for drought tolerance.
While root water status within the HFDI treatment was con-
sistent in sunflower, individuals were highly variable in poplar
as the higher CV for root and pre-dawn water potential indi-
cates. The ratio between leaf area and root weight explained
part of this variability in HFDI plants (Fig. 7B), which in
turn determined water uptake (Fig. 7A), but this ratio had
no impact in D/RW plants. Thus HFDI plants with a high
leaf area to upper root ratio had similar soil moisture in the
upper layer and root water status to D/RW, while low ratio
plants were similar to WW plants. This suggests that using
this irrigation procedure to screen for drought tolerance,
when leaf area or root allocation differs between genotypes,
simply reflects morphological differences that affect soil water
content of the upper layers and therefore root water poten-
tial. Genotypes with lower leaf area and a higher proportion
of roots in the upper soil layers maintained higher transpira-
tion rates and shoot water potential under suboptimal soil
moisture, while in many drought scenarios, deep rooting
accounts for drought tolerance (Puértolas et al., 2014; Lynch
and Wojciechowski, 2015).
Fig. 7. Relationships between the leaf area to root dry weight ratio in the As expected, frequent irrigation was necessary to main-
0–7 cm upper soil layer (LA/RWupper) and total plant water uptake (A), bulk tain a pre-determined suboptimal soil water content in the
root water potential (ψbulkroot) (B), and soil gravimetric water content (θg) in pot (Fig. 1), which altered the vertical distribution of soil
that layer (C), and between plant water uptake and θg in the upper layer moisture within the soil column in contrast to pots at the
(D) for each of the two deficit irrigation treatment (HFDI, white symbols; D/
same overall water content (weight) from which water was
RW, black symbols) and genotypes (B, circles; S, triangles) at the end of
the experiment in Populus nigra. For both irrigation treatments, the fitted withheld (Figs 3A, 8A). The small volume of water added
linear regression line and the P-values of the regression are shown (P<0.05 in each event was presumably taken up by the upper part
in the ANCOVA; HFDI, dashed; D/RW, dotted). of the root system, preventing its drainage to basal soil
 Drought experiments in potted plants and water relations | 2421

layers. Moreover, as soil dries, its hydraulic conductivity was homogeneous (Table 1). Interactions between root and
drops sharply (Lobet et al., 2014), further preventing water soil moisture distribution determine spatial patterns of water
movement from upper to lower soil layers. Since deeper uptake (Lobet et al., 2014), along with species- or genotype-
roots depleted soil water that was not replaced by irrigation, specific traits such as hydraulic architecture (Draye et al.,
deeper soil layers became drier in the HFDI treatment (Figs 2010), in a feedback mechanism that influences soil moisture
3A, 8A). This effect was more pronounced in Populus, where distribution. Together with the potential impact of genotypic
root weight was higher in the basal layer, than in sunflower, differences in biomass allocation (Table 1) on the creation of
with more roots in the upper part (Table 1). Soil moisture soil moisture gradients detailed above, this could obscure the
distribution in HFDI plants differed from that observed in results of species or genotype screening for water use strate-
plants from which water was withheld. Species differences in gies when applying high-frequency deficit irrigation to main-

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
soil water distribution within this treatment (D/RW) may be tain low soil water content in phenotyping platforms. As
partly explained by root distribution. In sunflower (higher an example, when comparing the drought responses of two
root weight in the upper part), soil moisture tended to be Populus genotypes using this irrigation approach, [X-ABA]
higher in the bottom in opposition to HFDI, while in pop- increased coincident with lower ψpd and lower transpiration
lar (higher root allocation at the bottom) soil water content in the genotype with higher leaf area (Chen et al., 1997).

Fig. 8. Soil gravimetric water content (θg) (A), root water potential (ψroot) (B), and ABA concentration in roots ([ABA]root) (C) in Populus nigra in different
irrigation treatments and soil layers (0–7 cm, white bars; 7–14 cm, patterned bars; 14–21 cm, black bars). Data are means ±SE of 14 replicates for HFDI
and D/RW, and 6 for WW, except for ψroot, where n=7 for HFDI and D/RW, and n=3 for WW. P-values for irrigation, genotype, layer, and their two-way
interactions in the repeated measures ANOVA are shown for each variable (no triple interaction was statistically significant). Different lower case letters
denote significant differences between depth×irrigation treatment combinations, while upper case letters denote differences of the average across depths
between irrigation treatments (Tukey, P<0.05).

Fig. 9. Graphic representation of the common main physiological effects of high-frequency deficit irrigation (HFDI) compared with optimal irrigation (WW)
and withholding water (D/RW) observed in this experiment. The relative effect is represented by the size of the ovals and, for gs and [X-ABA], arrows. The
effect on moisture in different soil layers is represented by different grey tones and textures (light grey>textured light grey>textured dark grey>dark grey).
[X-ABA], ABA concentration in xylem sap; [ABA]root, root ABA concentration averaged across the whole root system; ψroot/pd, root (or pre-dawn) water
potential; gs, stomatal conductance; θwhole pot, whole-pot soil water content. (This figure is available in colour at JXB online.)
2422 | Puértolas et al.

According to our results, that could not be interpreted as The light levels under which the experiments were con-
superior drought tolerance of this genotype but instead a ducted (400 µmol m–2 s–1) may have attenuated the effects of
consequence of faster water depletion. the two drought treatments on stomatal conductance, as the
Pronounced soil moisture gradients greatly affected effect of water deficit on gs is magnified at higher light inten-
root ABA accumulation, and this impact varied with spe- sities (Gimenez et al., 1992; Yin et al., 2006). Nevertheless,
cies. In H. annuus, local ABA accumulation in response to both treatments decreased gs or plant transpiration rate,
soil drying was much lower when a wet layer was present which could not be uniquely related to shoot water status or
(Fig. 4A), as in Phaseolus vulgaris, which was attributed xylem or leaf ABA concentration in both species (Figs 2, 5,
to redistribution of water within the root (Puértolas et al., 6B). On the contrary, they followed the same pattern across
2013). However, in poplar, [ABA]root responded to local soil irrigation treatments as ψbulkroot or ψpd in both species, with

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
drying more similarly in both drought treatments (Fig. 4B). intermediate values of gs or transpiration rate in HFDI plants
Although water redistribution from upper to lower roots compared with WW (higher) and D/RW (lower) explained by
driven by root water potential gradients is well documented intermediate values of ψpd or ψbulkroot (Fig. 2B, C and 5B, C,
(Burgess et al., 2001), the predominant pathways of this respectively). However, treatment variation in the rela-
water movement are not well known. However, assuming tionship between ψbulkroot and transpiration rate in poplar
similarity with the normal upward water flow, decreased (Fig. 6A) suggests decoupling between root water status and
radial conductivity in the endodermis might reduce the stomatal aperture, probably due to higher leaf ABA levels in
extent of the redistribution by limiting the reverse flow of HFDI plants of this species. Nevertheless, the coincidence
water from the xylem vessel to the cortex. The higher ABA between root water status and water use suggests the exist-
accumulation and lower root water potential in the lower ence of a root-sourced signal controlled by overall root water
layer of P. nigra might be explained by higher suberization status and not directly related to [X-ABA], which regulates
of the endodermis of woody compared with herbaceous stomatal aperture. Several candidates have been identified
species (Steudle, 2000), which may have impeded water including changes in xylem pH and other hormones or ions
redistribution within the root. Regardless of ABA accumu- (Davies et al., 2002; Ernst et al., 2010). Recent reports also
lation patterns, ABA export from roots in the dry low layers suggest that drought-induced suppression of strigolactone
to the shoots might be low (Boyle, 2015), in agreement with synthesis increases stomatal sensitivity to ABA (Visentin
the predictions of models explaining root to shoot ABA et al., 2016). The nature of this chemical long-distance signal
signalling in heterogeneous soil as a function of both ABA has attracted debate, and different mechanisms of its effect in
accumulation and the distribution of water uptake within the plant and the interaction with hydraulic signal have been
the rootzone (Dodd et al., 2008b; Puértolas et al., 2016), proposed (Tardieu, 2016). The soil moisture gradients gener-
as confirmed by the low [X-ABA] of HFDI plants (Figs ated with HFDI in species where shoot water status is not
2D, 5D). As observed previously (Khalil and Grace, 1993; altered by soil drying (such as tomato or poplar) might be
Puértolas et al., 2013), root ABA accumulation averaged an appropriate system to study stomatal regulation by non-
across the whole root system and xylem ABA concentration ABA root-sourced signals and the interaction with hydraulic
followed the same pattern across irrigation systems in both signals since the existence of a wet layer attenuates the ABA
species (Table 3, Fig. 9). signal while the dry soil still decreases stomatal conductance.

Table 3. Effect of high-frequency deficit irrigation (HFDI) on different variables compared with the effect of optimal irrigation (WW) and
withholding water (D/RW) in three species

Variable Treatment Helianthus annuus Populus nigra Solanum lycopersicum

[X-ABA] WW NS NS NS
D/RW +290% +130% +306%
[ABA]root WW –45% –29% NS
D/RW +63% +28% +249%
a
ψroot/pd WW +61% +80%
D/RW –28% –157% –285%
gs/Tr WW +56% +74% +136%
D/RW –24% –53% –55%

Data from H. annuus and P. nigra are from the present study, while for S. lycopersicum the data were extracted from Boyle (2015), where
watering with 100% (WW) and 50% of the potential evapotranspiration applied either daily (HFDI) or every 3 d (D/RW) were compared.
[X-ABA], ABA concentration in shoot (H. annuus, P. nigra) or leaf (S. lycopersicum) xylem sap; [ABA]root, ABA concentration in root tissue
averaged across the whole root; ψroot/pd, root (P. nigra, S. lycopersicum) or pre-dawn leaf (H. annuus) water potential; gs/Tr, stomatal
conductance (H. annuus, S. lycopersicum) or transpiration rate (P. nigra).
Values shown represent the percentage change in each treatment compared with the HFDI according to the formula: Change=100×[(Treat–
HFDI)/HFDI], where Treat is the mean value of the considered variable for that irrigation treatment and HFDI the mean value for the HFDI
treatment. Therefore, positive values indicate that the treatment was greater than the HFDI treatment, while negative values indicate that the
treatment was less than the HFDI treatment. NS, non-significant differences between the treatment and HFDI.
a
It was not possible to calculate ψroot for WW, since positive pressure was observed in all plants.
 Drought experiments in potted plants and water relations | 2423

Moreover, this irrigation approach could be better suited to Burgess SSO, Adams MA, Turner NC, White DA, Ong CK. 2001. Tree
roots: conduits for deep recharge of soil water. Oecologia 126, 158–165.
screen for tolerance to specific drought scenarios, where large
Chaves M, Davies B. 2010. Drought effects and water use efficiency:
soil moisture gradients are present and genotypic variability improving crop production in dry environments. Functional Plant Biology
in non-ABA root-sourced chemical or hydraulic signals are 37, III–VI.
important. For example, this approach could help discrimi- Chen S, Wang S, Altman A, Hüttermann A. 1997. Genotypic variation
nate the most drought tolerant within a selection of deep- in drought tolerance of poplar in relation to abscisic acid. Tree Physiology
17, 797–803.
rooted genotypes. Therefore, even though our results indicate
Davies WJ, Wilkinson S, Loveys B. 2002. Stomatal control by chemical
certain limitations to the use of phenotyping platforms to signalling and the exploitation of this mechanism to increase water use
screen for drought tolerance, they still can be very useful to efficiency in agriculture. New Phytologist 153, 449–460.
understand the physiological effects of different irrigation Dodd IC, Egea G, Davies WJ. 2008a. Abscisic acid signalling when soil

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
procedures. moisture is heterogeneous: decreased photoperiod sap flow from drying
roots limits abscisic acid export to the shoots. Plant, Cell and Environment
31, 1263–1274.
Conclusions Dodd IC, Egea G, Davies WJ. 2008b. Accounting for sap flow from
different parts of the root system improves the prediction of xylem ABA
When long-term experiments to assess crop drought toler- concentration in plants grown with heterogeneous soil moisture. Journal of
ance maintain constant, suboptimal whole-pot soil water Experimental Botany 59, 4083–4093.
content via high irrigation frequency, large soil moisture gra- Draye X, Kim Y, Lobet G, Javaux M. 2010. Model-assisted integration
dients are created within the pot, with wet upper and very dry of physiological and environmental constraints affecting the dynamic and
spatial patterns of root water uptake from soils. Journal of Experimental
lower layers. This treatment consistently limited root ABA Botany 61, 2145–2155.
accumulation (Fig. 4) and suppressed long-distance ABA sig- Einhorn TC, Caspari HW, Green S. 2012. Total soil water content
nalling (same [X-ABA] as WW plants but much lower than in accounts for augmented ABA leaf concentration and stomatal regulation
D/RW plants; Fig. 9; Table 3). Thus gs was better related to of split-rooted apple trees during heterogeneous soil drying. Journal of
Experimental Botany 63, 5365–5376.
ψroot than to [X-ABA], suggesting that another root-sourced
Ernst L, Goodger JQ, Alvarez S, et al. 2010. Sulphate as a xylem-
chemical signal induced partial stomatal closure. Shoot water borne chemical signal precedes the expression of ABA biosynthetic genes
potential did not seem responsible, since ψshoot responses var- in maize roots. Journal of Experimental Botany 61, 3395–3405.
ied between species and were not related to gs (Figs 2, 5). All Furbank RT, Tester M. 2011. Phenomics—technologies to relieve the
these physiological responses depended on moisture content phenotyping bottleneck. Trends in Plant Science 16, 635–644.
of the uppermost soil layer which in turn was influenced by Gimenez C, Mitchell VJ, Lawlor DW. 1992. Regulation of
photosynthetic rate of two sunflower hybrids under water stress. Plant
the plant water uptake rate (Fig. 7), which might invalidate Physiology 98, 516–524.
the use of the high-frequency deficit irrigation in genotype Granier C, Aguirrezabal L, Chenu K, et al. 2006. PHENOPSIS, an
screening for drought tolerance. automated platform for reproducible phenotyping of plant responses to
soil water deficit in Arabidopsis thaliana permitted the identification of an
accession with low sensitivity to soil water deficit. New Phytologist 169,
Acknowledgements 623–635.
Hu H, Xiong L. 2014. Genetic engineering and breeding of drought-
The research reported here was conducted in WATBIO (Development of resistant crops. Annual Review of Plant Biology 65, 715–741.
improved perennial non-food biomass and bioproduct crops for water-
stressed environments) which is a collaborative research project funded from Khalil AAM, Grace J. 1993. Does xylem sap ABA control the stomatal
the European Union’s Seventh Programme for research, technological devel- behavior of water-stressed sycamore (Acer pseudoplatanus L) seedlings?
opment, and demonstration under grant agreement no. 311929. Journal of Experimental Botany 44, 1127–1134.
Lawlor DW. 2013. Genetic engineering to improve plant performance
under drought: physiological evaluation of achievements, limitations, and
possibilities. Journal of Experimental Botany 64, 83–108.
References
Lobet G, Couvreur V, Meunier F, Javaux M, Draye X. 2014. Plant
Baginsky S, Hennig L, Zimmermann P, Gruissem W. 2010. Gene water uptake in drying soils. Plant Physiology 164, 1619–1627.
expression analysis, proteomics, and network discovery. Plant Physiology Lynch JP, Wojciechowski T. 2015. Opportunities and challenges in the
152, 402–410. subsoil: pathways to deeper rooted crops. Journal of Experimental Botany
Blum A. 2011. Plant breeding for water limited environments. New York: 66, 2199–2210.
Springer Publishing. McAdam SAM. 2015. Physicochemical quantification of abscisic acid
Blum A. 2014. Genomics for drought resistance—getting down to earth. levels in plant tissues with an added internal standard by ultra-performance
Functional Plant Biology 41, 1191–1198. liquid chromatography. Bio Protocol 5, e1599.
Blum A. 2015. Towards a conceptual ABA ideotype in plant breeding for Neilson EH, Edwards AM, Blomstedt CK, Berger B, Møller BL,
water limited environments. Functional Plant Biology 42, 502–513. Gleadow RM. 2015. Utilization of a high-throughput shoot imaging
Boyle RKA. 2015. Effects of deficit irrigation frequency on plant growth, system to examine the dynamic phenotypic responses of a C4 cereal crop
water use and physiology of Pelargonium×hortorum and tomato (Solanum plant to nitrogen and water deficiency over time. Journal of Experimental
lycopersicum L. cv. Ailsa Craig). PhD Thesis, Lancaster University. Botany 66, 1817–1832.
Boyle RK, McAinsh M, Dodd IC. 2016a. Daily irrigation attenuates Passioura JB. 2012. Phenotyping for drought tolerance in grain crops:
xylem abscisic acid concentration and increases leaf water potential of when is it useful to breeders? Functional Plant Biology 39, 851–859.
Pelargonium × hortorum compared with infrequent irrigation. Physiologia Pereyra-Irujo GA, Gasco ED, Peirone LS, Aguirrezabal LAN. 2012.
Plantarum 158, 23–33. GlyPh: a low-cost platform for phenotyping plant growth and water use.
Boyle RK, McAinsh M, Dodd IC. 2016b. Stomatal closure of Functional Plant Biology 39, 905–913.
Pelargonium × hortorum in response to soil water deficit is associated with Poorter H, Bühler J, van Dusschoten D, Climent J, Postma JA.
decreased leaf water potential only under rapid soil drying. Physiologia 2012. Pot size matters: a meta-analysis of the effects of rooting volume on
Plantarum 156, 84–96. plant growth. Functional Plant Biology 39, 839–850.
2424 | Puértolas et al.
Poorter H, Fiorani F, Pieruschka R, Wojciechowski T, van der Putten Sreenivasulu N, Harshavardhan VT, Govind G, Seiler C, Kohli A.
WH, Kleyer M, Schurr U, Postma J. 2016. Pampered inside, pestered 2012. Contrapuntal role of ABA: does it mediate stress tolerance or
outside? Differences and similarities between plants growing in controlled plant growth retardation under long-term drought stress? Gene 506,
conditions and in the field. New Phytologist 212, 838–855. 265–273.
Puértolas J, Alcobendas R, Alarcón JJ, Dodd IC. 2013. Long-distance Steudle E. 2000. Water uptake by plant roots: an integration of views.
abscisic acid signalling under different vertical soil moisture gradients Plant and Soil 226, 45–56.
depends on bulk root water potential and average soil water content in the Tardieu F. 2012. Any trait or trait-related allele can confer drought
root zone. Plant, Cell and Environment 36, 1465–1475. tolerance: just design the right drought scenario. Journal of Experimental
Puértolas J, Ballester C, Elphinstone ED, Dodd IC. 2014. Two potato Botany 63, 25–31.
(Solanum tuberosum) varieties differ in drought tolerance due to differences Tardieu F. 2016. Too many partners in root–shoot signals. Does hydraulics
in root growth at depth. Functional Plant Biology 41, 1107–1118. qualify as the only signal that feeds back over time for reliable stomatal
Puértolas J, Dodd IC, Conesa MC. 2016. An empirical model predicting control? New Phytologist 212, 802–804.

Downloaded from https://academic.oup.com/jxb/article/68/9/2413/3737164 by Szkola Glowna Gospodarstwa Wiejskiego user on 31 May 2023
xylem sap ABA concentration from root biomass and soil moisture distribution Tardieu F, Simonneau T. 1998. Variability among species of stomatal
in plants under partial root-zone drying. Acta Horticulturae 1112, 147–153. control under fluctuating soil water status and evaporative demand:
Quarrie SA, Whitford PN, Appleford NE, Wang TL, Cook SK, Henson modelling isohydric and anisohydric behaviours. Journal of Experimental
IE, Loveys BR. 1988. A monoclonal antibody to (S)-abscisic acid: its Botany 49, 419–432.
characterisation and use in a radioimmunoassay for measuring abscisic Tisné S, Serrand Y, Bach L, et al. 2013. Phenoscope: an automated
acid in crude extracts of cereal and lupin leaves. Planta 173, 330–339. large-scale phenotyping platform offering high spatial homogeneity. The
Saradadevi R, Bramley H, Siddique KHM, Edwards E, Palta JA. Plant Journal 74, 534–544.
2014. Contrasting stomatal regulation and leaf ABA concentrations in Visentin I, Vitali M, Ferrero M, et al. 2016. Low levels of strigolactones
wheat genotypes when split root systems were exposed to terminal in roots as a component of the systemic signal of drought stress in tomato.
drought. Field Crops Research 162, 77–86. New Phytologist 212, 954–963.
Segal E, Ben-Gal A, Shani U. 2006. Root water uptake efficiency under Yin CY, Berninger F, Li CY. 2006. Photosynthetic responses of Populus
ultra-high irrigation frequency. Plant and Soil 282, 333–341. przewalski subjected to drought stress. Photosynthetica 44, 62–68.