Journal of Experimental Botany, Page 1 of 15
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
Water stress drastically reduces root growth and inulin yield
in Cichorium intybus (var. sativum) independently of
B. Vandoorne1,4, A.-S. Mathieu1, W. Van den Ende3, R. Vergauwen3, C. Pe ´rilleux2, M. Javaux4,5and S. Lutts1,*
1Groupe de Recherche en Physiologie Ve ´ge ´tale (GRPV), Earth and Life Institute – Agronomy (ELI-A), Universite ´ catholique de Louvain,
5 (Bte L 7.07.13) Place Croix du Sud, 1348 Louvain-la-Neuve, Belgium
2Laboratory of Plant Physiology, Department of Life Sciences, University of Lie `ge, B22 Sart Tilman, 27 Boulevard du Rectorat, B-4000
Lie `ge, Belgium
3Laboratory of Molecular Plant Physiology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, Kasteelpark Arenberg
31, 3001 Leuven-Heverlee, Belgium
4Earth and Life Institute – Environmental Sciences (ELI-E), Universite ´ catholique de Louvain, 2 (Bte 2) Place Croix du Sud, 1348
5Agrosphere (IBG-3), Institut fu ¨r Bio- und Geowissenschaften - Forschungszentrum Juelich GmBH, Juelich, Germany
* To whom correspondence should be addressed. E-mail: email@example.com
Received 1 December 2011; Revised 28 February 2012; Accepted 1 March 2012
Root chicory (Cichorium intybus var. sativum) is a cash crop cultivated for inulin production in Western Europe. This
plant can be exposed to severe water stress during the last 3 months of its 6-month growing period. The aim of this
study was to quantify the effect of a progressive decline in water availability on plant growth, photosynthesis, and
sugar metabolism and to determine its impact on inulin production. Water stress drastically decreased fresh and dry
root weight, leaf number, total leaf area, and stomatal conductance. Stressed plants, however, increased their
water-use efficiency and leaf soluble sugar concentration, decreased the shoot-to-root ratio and lowered their
osmotic potential. Despite a decrease in photosynthetic pigments, the photosynthesis light phase remained
unaffected under water stress. Water stress increased sucrose phosphate synthase activity in the leaves but not in
the roots. Water stress inhibited sucrose:sucrose 1-fructosyltransferase and fructan:fructan 1 fructosyltransferase
after 19 weeks of culture and slightly increased fructan 1-exohydrolase activity. The root inulin concentration,
expressed on a dry-weight basis, and the mean degree of polymerization of the inulin chain remained unaffected by
water stress. Root chicory displayed resistance to water stress, but that resistance was obtained at the expense of
growth, which in turn led to a significant decrease in inulin production.
Key words: Cichorium intybus, drought, growth, inulin, photosynthesis, root chicory, sugar metabolism, water deficit, water stress.
Cichorium intybus var. sativum is a biannual crop cultivated
mainly in Western Europe and to a lesser extent in other
parts of the world to produce inulin, which is a linear
b(2,1)-type fructan that is widely used as a prebiotic agent
with antioxidant properties (Stoyanova et al., 2011). Inulins
selectively stimulate ’good bacteria’ (such as Bifidobacteria
and Lactobacillae) in the colon, contributing to overall good
health and helping disease prevention (Roberfroid and
Delzenne, 1998). During the growing season (first year), inulin
is stored in the chicory tap roots as a reserve component.
Abbreviations: A, net CO2assimilation rate; DP, degree of polymerization; DW, dry weight; E, instantaneous transpiration rate; EDTA, ethylenediaminetetra-acetic acid;
1-FEH, fructan 1-exohydrolase; 1-FFT, fructan:fructan 1 fructosyltransferase; FW, fresh weight; HPAEC-PAD, high-performance anion-exchange chromatography with
pulsed amperometric detection; MDA, malondialdehyde; NPQ, non-photochemical quenching; uPSII, photosystem II efficiency; qp, photochemical quenching; SPS,
sucrose phosphate synthase; 1-SST, sucrose:sucrose 1-fructosyltransferase; SuSy, sucrose synthase.
ª The Author . 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Journal of Experimental Botany Advance Access published May 10, 2012
by guest on May 28, 2012
Under natural conditions, this reserve allows chicory to
survive winter and accomplish its reproductive cycle during
the second year (Van Laere and Van den Ende, 2002).
Fructans are also known to protect cells from desiccation
during water stress, which is in line with their role as
membrane stabilizers (Hendry, 1993; Valluru and Van den
Ende, 2008). Water stress leads to an increased fructan
content in numerous plant species (Spollen and Nelson,
1994; Volaire and Lelie `vre, 1997; Kerepesi et al., 1998) and
in bryophytes (Marschall et al., 1998). According to Pilon-
Smits et al. (1995), transgenic tobacco containing fructans
exhibits an improved resistance to water deficit compared
with wild-type tobacco.
Inulin synthesis isinitiated
1-fructosyltransferase (1-SST, EC 184.108.40.206), which catalyses
the transfer of a fructose moiety between two sucrose
molecules to produce glucose and the trisaccharide 1-kestose.
Fructan:fructan 1-fructosyltransferase (1-FFT, EC 220.127.116.11)
transfers fructan moieties from and to 1-kestose or larger
fructans, resulting in the formation of oligofructose and
inulin with a higher degree of polymerization (DP). Con-
versely, inulin degradation involves a sequential removal of
fructose units catalysed by fructan 1-exohydrolases (1-FEHs;
EC 18.104.22.168) (Van Laere and Van den Ende, 2002). The
mean DP of the inulin chain is an important component of
root chicory production, with a higher DP being more
suitable for industrial applications (Wilson et al., 2004).
According to the Intergovernmental Panel on Climate
Change, scenarios with drier summers are expected to occur
in Western Europe in the next few decades (IPCC, 2007). In
several parts of the world, root chicory is now cultivated
under irrigation, but a better knowledge of its behaviour
under water stress conditions should allow the minimal
water requirements of this species to be defined and thus
allow its culture to be extended to regions where irrigation
is not technically feasible or economically justified. Although
responses to low temperature have been extensively studied
in this species, mainly with respect to vernalization (Dielen
et al., 2005; Devacht et al., 2009; Mingeot et al., 2009), the
impact of water stress has received less attention. De Roover
et al. (2000) studied the impact of water stress at the seedling
stage and demonstrated that water shortage increased
glucose, fructose, and sucrose concentrations in the roots
and leaves of stressed plants, leading to increased fructan
concentrations in the roots. Using a field approach, Monti
et al. (2005) demonstrated that water shortage had only
a limited impact on yield, leaf photosynthetic capacity was
poorly affected by water availability, and fructan chain
length was not affected by the water regime. Only moderate
water stress was applied in this study, and the activities of
enzymes involved in inulin metabolism were not quantified.
Recently, the severity and timing of drought were reported to
strongly influence the sequence of plant reactions to water
shortage (Muller et al., 2011; Skirycz et al., 2011).
Water stress that impacts photosynthesis in root chicory
has been suggested to mainly be related to non-stomatal
effects (Monti et al., 2005). Although data on the impact of
drought on ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco; EC 22.214.171.124) activity in this species are still missing,
this enzyme was shown to be drastically affected in numerous
plant species exposed to water shortages (Parry et al.,
2002). Although a decline in Rubisco activity is expected
to decrease sugar synthesis, water stress often paradoxi-
cally increases sucrose content through the stimulation of
sucrose phosphate synthase (SPS; EC 126.96.36.199) (Fu et al.,
2010). Therefore, the precise sugar status in water-stressed
tissues could be considered to be the ultimate consequence
of several interacting factors related to growth inhibition,
CO2 fixation, and enzyme activities involved in sucrose
synthesis on the one hand (SPS), and sucrose breakdown
(acid and neutral invertases; EC 188.8.131.52) on the other.
Sucrose synthase (SuSy; EC 184.108.40.206) catalyses a reversible
reaction, but it is usually involved in sucrose breakdown and
generates UDP-glucose and fructose.
The soluble sugar concentration may directly influence
inulin concentration in root chicory: sucrose was reported
to increase the expression of the gene encoding 1-SST,
leading to an increase in the corresponding enzyme activity
(De Roover et al., 2000). According to Van Laere and Van
den Ende (2002), vacuolar 1-SST may itself be involved in
sink strength determination, thereby contributing to yield
maintenance under stress conditions. Conversely, an in-
crease in sucrose was reported to inhibit FEH activities, at
least for some isoforms (Verhaest et al., 2007).
The present study was undertaken to evaluate the
physiological and yield impacts of water stress on root
chicory. Plant behaviour was analysed during and after
a significant decrease in the soil water content. This kinetics
approach aimed to identify the main morphophysiological
parameters contributing to the deleterious impact of water
stress on inulin production in relation to enzymatic activities
involved in sugar metabolism.
Materials and methods
Plant material and growth culture
Seeds of C. intybus var. sativum (purified line issued from cultivar
Crescendo and kindly provided by the Cosucra Group, Warcoing SA
division, Chicoline, Belgium) were sown in columns that were 55 cm
long and 25 cm in diameter. The 21 columns were filled with dry
yellow sand, and 0.5 dm3of loam (loam for professionals; DCM
N.V., Grobbendonk, Belgium) was added at the top. The mean
temperature during the growing season was 23 ?C during the 16-h day
and 20 ?C during the 8-h night. The mean relative humidity in the air
was 75%, and the light intensity was 135 lmol m?2s?1(six Philips
HPI-T lamps; 400 W) at the top of the canopy. Four days after
germination only one plant was maintained in each column. Plants
were provided with a nutrient solution of pH 5.4 and electrical
conductivity of 963 lS cm?1containing the following nutrient
concentrations: 250 lM NH4NO3, 890 lM Ca(NO3)2.4H2O, 990 lM
KNO3, 515 lM KH2PO4, 244 lM MgSO4.7H2O, 0.415 lM
MnSO4.5H2O, 6.45 lM H3BO3, 0.161 lM CuSO4.5H2O, 0.0125 lM
(NH4)6Mo7O24.4H2O, 0.697 lM ZnSO4.7H2O, and 10.12 lM
Fe-EDDHA [ethylenediamine-N,N’-bis(2-hydroxyphenylacetic acid)].
To avoid evaporation from the soil surface, the top of each column
was covered with a perforated Plexiglas plate, allowing only the basal
part of the rosette to emerge.
During the experiment, four harvests were performed at 7, 13,
19, and 25 weeks after sowing. Three plants per treatment were
collected at each harvest. The drought stress started 12 weeks after
2 of 15 | Vandoorne et al.
by guest on May 28, 2012
sowing. At this time, plants were distributed into two groups:
control and drought-stressed plants. Water was supplied to the
control plants to maintain the volumetric water content (hv)
between 11 and 19% (close to field capacity: hv¼ 15%) throughout
the culture period, whereas one-fifth of this volume was supplied
to the stressed plants, which were permanently exposed to a
substrate with less than 3% of its volumetric water content (close
to the wilting point: hv¼ 2%) (Fig. 1).
The total leaf area was measured at each harvest for three plants
per treatment using a Leaf Area Meter AM300 (ADC Bioscientific,
Hoddesdon, Hertfordshire, UK). The mean duration required for
the macroscopic appearance of a new leaf was calculated by
dividing the number of days between two measurements by the
number of leaves appearing between the measurement times.
Plant water status
The water contents of leaves and roots were measured after drying
the samples in an oven for 72 h at 70 ?C. The leaf water potential
(ww) was measured at each harvest at mid-day (between 12 a.m.
and 2 p.m.) on the fifth and sixth unfolded leaves using a Scho-
lander pressure chamber, considering the youngest unfolded leaf of
the rosette as no. 1. The osmotic potential (wsraw) was measured
on a portion of the second fully unfolded leaf with a Vapour
Pressure Osmometer 5520 (Wescor, Logan, CT, USA) and adjusted
to the water content of the control plants according to Lefe `vre et al.
(2009): ws ¼ wsraw*(WCs/WCcontrol), where WCscorresponds to the
water content of the stressed plants and WCcontrol to the water
content of the control plants.
The stomatal conductance (gs) was measured using an AP4
system (Delta-T Devices, Cambridge, UK) on the first unfolded
leaf and four other randomly selected leaves halfway through the
Chlorophyll fluorescence-related parameters were measured for
five plants per treatment by the Fluorescence Monitoring System
II (Hansatech Instruments, Norfolk, UK) on the second and third
unfolded leaves after dark-adaption for 30 min. After a saturating
pulse (18 000 lmol m?2s?1) was sent to the leaf, the leaf was
exposed to a constant intensity of actinic light (600 lmol m?2s?1)
for 3 min, followed by a second saturating pulse of 18 000 lmol
m?2s?1. Photosystem II efficiency (uPSII), non-photochemical
quenching (NPQ), and photochemical quenching (qp) were esti-
mated according to Maxwell and Johnson (2000).
Chlorophyll (Chl a and Chl b) and total carotenoid (xantho-
phylls + b-carotene) concentrations were quantified for three
plants per treatment on the sixth fully unfolded leaf in the rosette.
Samples [;150 mg fresh weight (FW)] were ground in the dark in
8 ml of 80% acetone and centrifuged at 1000 g for 10 min at 4 ?C.
The absorbance of the sample was read at three different
wavelengths (663.2, 646.8, and 470 nm) using a spectrophotometer
(DU 640, Beckman Coulter, South Pasadana, CA, USA). Each
measurement was repeated three times. The pigment concentra-
tions were calculated according to Lichtenthaler (1987).
Gas exchange was recorded with an infrared gas analyser (LCA4
8.7; ADC Bioscientific, Hoddesdon, Hertfordshire, UK) using a
PLC Parkinson leaf cuvette on intact leaves for 1 min (20 records
min?1) and an air flow of 300 ml min?1. Air taken in the green-
houses was sent to a chamber into which a leaf portion of 6.25 cm2
was introduced. The net CO2assimilation rate (A) and instanta-
neous transpiration rate (E) were estimated on the second and
third fully unfolded leaves. Five plants were measured for each
treatment, and all measurements were performed around midday
(between 12 a.m. and 2 p.m.).
For determination of Rubisco (EC 220.127.116.11), fresh matter was
collected on the third, fourth, and fifth unfolded leaves for three plants
per treatment, quickly frozen in liquid nitrogen and homogenized in
4 ml of extraction buffer [100 mM Tris/HCl, pH 7.8, containing 0.4
mM ATP, 10 mM MgCl2, 1 mM ethylenediaminetetra-acetic acid
(EDTA), 0.1% (v/v) Triton X-100, 12.5% (w/v) glycerol, 15 mM
mercaptoethanol, and 30 mg polyvinyl polypyrrolidone]. The extract
was centrifuged for 30 s at 10 000 g and 4 ?C. To measure the initial
activity, 30 ll of this extract was rapidly added to 970 ll of a reaction
buffer consisting of 50 mM Hepes/KOH, pH 8.0, 20 mM MgCl2, 10
mM KCl, 1 mM EDTA, 5 mM DTT, 2.5 mM ATP, 5 mM
phosphocreatine, 0.2 mM NADH, 0.6 mM ribulose 1,5-bisphosphate,
10 mM NaHCO3, 6 U ml?1phosphoglycerate kinase (Sigma
ALdrich, St Louis, MO, USA), 6 U l?1glycerate 3-phosphate
dehydrogenase (Sigma), and 20 U ml?1phosphocreatine kinase
(Sigma). The oxidation of NADH was determined during a 3 min
period at 25 ?C by measuring the difference between the absor-
bances at 340 and 400 nm. The total activity was quantified acco-
rding to the same procedure after an activation period of 10 min in
20 mM MgCl2 and 10 mM NaHCO3. The activation ratio was
Fig. 1. Volume of nutrient solution supplied to each column (in l week?1) (A) and soil volumetric water content (B) during the 25 weeks of
plant culture for C. intybus exposed to well-watered (control) and drought (drought) conditions. Filled symbols indicate control plants and
open symbols indicate drought-stressed plants. In Fig. 1B, each value is the mean of 3 replicates and vertical bars are the standard
errors. The stars indicate the presence of a statistical difference between the treatments (*: P< 0.05; **: P< 0.01 and ***: P< 0.001).
Water stress reduces root growth and inulin yield | 3 of 15
by guest on May 28, 2012
calculated by dividing the initial activity by the total activity accor-
ding to Du et al. (1996). Two replicates were made for each sample.
Leaf malondialdehyde concentration
The level of lipid peroxidation was measured as the 2-thiobarbituric
(MDA), following the modified method of Heath and Packer
(1968). Leaf portions (250 mg FW) were ground in liquid nitrogen
with 5 ml of a solution of 5% trichloracetic acid and 1.25% glycerol.
The homogenate was centrifuged at 6700 g for 10 min at 4 ?C and
then filtered through Whatman paper no. 1 filter paper. Then 2 ml
of the supernatant was mixed with 2 ml 0.67% thiobarbituric acid.
The samples were incubated at 100 ?C for 30 min and immediately
transferred to ice for 5 min, followed by centrifugation at 6700 g for
1 min at 4 ?C. The supernatant absorbance was read at 532 nm, and
values corresponding to non-specific absorption (600 nm) were
subtracted. MDA concentration was calculated using its molar
extinction coefficient (e ¼ 155 mM?1cm?1).
Portions of the third, fourth, and fifth unfolded leaves (;300 mg
FW) collected from three plants per treatment were ground in
liquid nitrogen, mixed with 7 ml of 70% ethanol (w/v) for 5 min on
ice and centrifuged at 6700 g for 10 min at 4 ?C. After reacting
200 ll of the supernatant with 1 ml of an anthrone solution (0.5 g
anthrone, 250 ml 95% H2SO4, and 12.5 ml distilled water), the
absorbance was read at 625 nm (spectrophotometer UV-1800,
Shimadzu, Kyoto, Japan) to quantify the total soluble sugars
according to Yemm and Willis (1954). A calibration curve was
established using glucose as the standard.
The remaining supernatants were dried in a thermomixer using
nitrogen (45 min at 50 ?C), and sugars were derivatized using
200 ll of an oxymation solution (hydroxylamine chlorhydrate
dissolved in a pyridine solution): 100 ll of hexamethyldisilazane
and 10 ll of trifluoroacetic acid were added to the samples. Mono-, di-,
and trisaccharide concentrations were specifically determined by
gas chromatography (Autosystem XL, MN Optima-5 Accent,
Perkin Elmer, Waltham, MA, USA; 30 m30.32 mm internal
diameter30.25 lm) using helium as a carrier gas at a flow rate of
1 ml min?1. The injector and flame ionization detector temper-
atures were 270 and 310 ?C, respectively. The oven temperature
was held at 120 ?C for 3 min and then programmed to 230 ?C at
3 ?C min?1. It was kept at this temperature for 12 min, then
re-programmed to 300 ?C at 20 ?C min?1and held for 15 min.
To extract sugars and enzymes from the roots, 30 g of root
material was ground in 30 ml of 50 mM Hepes/KOH, pH 7.5,
containing 100 mM KCl, 30 mM MgCl2, 10 mM NaHSO3, 2 mM
EDTA, 1 mM phenylmethylsulphonylfluoride, 1 mM mercaptoe-
thanol, and 0.1% Polyclar (Serva, Heidelberg, Germany). Then,
2 ml was incubated at 90 ?C for 15 min. After cooling at room
temperature, the extract was centrifuged at 3000 g for 5 min, and
700 ll of the supernatant were purified on a column with two ion-
exchange resins (1 ml Dowex 50 H+and 1 ml Dowex 1-acetate).
Finally, 25 ll of the neutralized fraction was analysed by high-
performance anion-exchange chromatography with pulsed ampero-
metric detection (HPAEC-PAD; Dionex, Sunnyvale, CA, USA) to
determine the sugar content after separation on a CarboPac? PA1
anion exchange column and detection by a pulsed anperometric
detector equipped with a gold electrode (potentials: E1: + 0.05 V;
E2: +0.6 V; E3: –0.8 V). The flow rate was 1 ml.min?1. The elution
conditions were 90 mM NaOH over a 60-min period. The column
was regenerated with 1 M NaOH for 10 min and equilibrated for
20 min after each run. Quantification was performed on the peak
areas using the external standards methods for glucose, fructose,
sucrose, 1-kestose and 1,1-nystose (Van den Ende et al., 1996). In
parallel, 25 ll of the neutralized fraction was mixed with 6 N HCl to
completely depolymerize all of the inulin into its single components
(glucose and fructose). The extract was analysed using the same
The mean DP is calculated according to a formula adapted from
DPmean¼Fhydrol? Ffree? Sfree
Ghydrol? Gfree? Sfreeþ 1
where Fhydroland Ghydrolare the concentrations of fructose (F) and
glucose (G) after hydrolysis with HCl, and Ffree, Gfree, and Sfreeare
the concentrations of free fructose, glucose, and sucrose (S).
The percentage of inulin in the root was estimated using
a formula adapted from Baert (1997):
Fhydrol? Ffree? Sfreeþ Ghydrol? Gfree? Sfree
1000 ? ð1 ? WCÞ
considering that each addition of one fructose unit to the inulin
chain induces the loss of one molecule of water (18 g mol?1).
Root 1-SST (EC 18.104.22.168), 1-FFT (EC 22.214.171.124), and 1-FEH (EC
126.96.36.199) activities were determined according to Van den Ende
et al. (1996) for three plants per treatment. After 5 ml of the
extraction buffer containing the extract was centrifuged for 2 min
at 10 000 g, two aliquots (2 ml each) were mixed with 4 ml of
saturated ammonium sulphate. After 30 minutes at 0 ?C, the pellet
was centrifuged at 40 000 g for 15 min and washed with
ammonium sulphate 80% (w/v). The pellet was centrifuged again
under the same conditions, suspended in 500 ll of 50 mM sodium
acetate buffer, pH 5.0, and then centrifuged for 2 min at 10 000 g.
The enzymes 1-SST and 1-FEH were incubated with their sub-
strate at 30 ?C (100 mM sucrose for 1-SST and 1% inulin for
1-FEH) for 0, 20, 40, and 60 min for 1-SST and 0, 40, 80, and 120
min for 1-FEH. The 1-FFT enzyme was incubated with 10 mM
1-kestose on ice for 0, 20, 40, and 60 min. The reactions were
stopped by incubating the samples at 95 ?C for 5 min. The enzyme
activities were determined by product quantification (1-kestose for
1-SST, 1,1-kestotetraose for 1-FFT, and fructose for 1-FEH) with
SPS (EC 188.8.131.52), SuSy (EC 184.108.40.206), and invertase (acid and
neutral; EC 220.127.116.11) activities were determined in the leaves and
roots. For this purpose, 700 mg of material was combined with
3.5 ml of extraction buffer (50 mM Hepes/KOH, pH 7.5,
containing 100 mM KCl, 20 mM MgCl2, 10 mM NaHSO3, 2 mM
EDTA, 1 mM phenylmethylsulphonylfluoride, 1 mM mercaptoe-
thanol, and 0.1% Polyclar) for leaves, and 5 g of material was
combined with 5 ml of the extraction buffer for roots. The extract
was then centrifuged at 10 000 g for 5 min at 4 ?C. Two aliquots
(1.5 ml each) were mixed with 6 ml of saturated ammonium
sulphate. After 30 min at 0 ?C, the pellet was centrifuged at 40 000
g for 15 min, washed with 80% (w/v) ammonium sulphate, and
centrifuged as before. The obtained pellet was then resuspended in
3 ml of 50 mM Hepes/KOH buffer, pH 7.5, containing 100 mM
KCl, 20 mM MgCl2, and 2 mM EDTA. SPS activity was
determined according to Huber et al. (1991). SuSy and invertase
activities were assessed according to King et al. (1997), with slight
modifications involving the use of a 3,5-dinitrosalicylic acid
solution (45 mM 3,5-dinitrosalicylic acid, 10% NaOH, and 1 M
potassium sodium tartrate) to stop the reaction after incubation.
The protein concentration in the extract was estimated accord-
ing to Bradford (1976) using bovine serum albumin as standard.
Statistical treatment of the data: At each harvest, three plants per
treatment were analysed, except in cases of non-destructive
physiological analysis, for which five plants per treatment were
used. Each experiment was repeated twice and exhibited similar
trends. Data presented hereafter are from one representative
experiment. The statistical analysis was performed using SAS
4 of 15 | Vandoorne et al.
by guest on May 28, 2012
software (SAS System for Windows, version 9.1). The normal
distributions of the data were analysed using a Shapiro–Wilk test.
When needed, the data were transformed to have a normal
Data were then subjected to an analysis of variance (ANOVA I)
considering the presence or absence of stress as a factor. The
statistical significance of the results was analysed by the Student–
Newman–Keuls test at the 5% level.
Drought drastically decreased the rate of leaf appearance in
the rosette throughout the stress period: the mean duration
required for the macroscopic appearance of a new leaf was
always higher in stressed plants than in controls (Fig. 2A).
The total leaf surface was lower in stressed plants than in
controls (Fig. 2B), as a result of not only the lower number
of leaves but also a strong decrease in the mean leaf area
(Fig. 2C). The total leaf surface decreased from week 13 to
week 19 due to stress-induced senescence leading to leaf
abscission (Fig. 2D). The specific leaf area was significantly
lower in stressed plants [148.9614.8 and 170.467.5 cm2g?1
dry weight (DW) after 19 and 25 weeks, respectively] than
in controls (257.8 610.6 and 222.6617.2 cm2g?1DW after
19 and 25 weeks, respectively). The mean root FW was also
lower in the stressed plants, with a weight decrease of more
than 50% at the end of the stress period compared with
controls (Fig. 2E). The shoot-to-root ratio decreased from
week 7 to week 19 and remained constant thereafter for the
controls. At the end of the experiment, this ratio was 2-fold
higher in the control plants compared with the stressed
plants (Fig. 2F), suggesting that shoot growth was more
inhibited than root growth under water shortage conditions.
The ratio of the total leaf area/root DW was more than
2-fold lower in stressed plants than in controls after 19 weeks
(8.661.1 in controls versus 3.560.5 in stressed plants) and
after 25 weeks (7.161.6 in controls versus 2.360.3 in
Plant water status
The water content (Fig. 3A) was lower in the roots than in
the leaves and remained higher in the control plants than in
the stressed plants. Among treatments, no significant
difference was recorded for the leaf water potential (ww)
(Fig. 3B). In contrast, both leaf and root osmotic potentials
(ws) were lower in the stressed plants than in the controls
(Fig. 3C, 3D). As far as root wsis concerned, the difference
was not significant at the end of the treatment. Drought had
a strong deleterious impact on the global stomatal conduc-
tance (gs) of chicory leaves, which was lower in the drought-
stressed plants than in the controls (Table 1).
Gas exchange and photosynthesis-related parameters
Notably, neither the instantaneous transpiration (E; Table 1)
nor the net photosynthesis (A; Table 1) of young leaves were
affected in response to drought. At week 19, net photosyn-
thesis was even higher in the stressed plants than in the
controls, while the difference between treatments remained
statistically non-significant for other periods of stress. Chloro-
phyll concentrations (Fig. 4A, 4B) decreased in the stressed
plants, but the ratio Chl a/Chl b remained unchanged
(Fig. 4C). A similar stress-induced decrease was observed for
carotenoids (Fig. 4D). Despite the recorded changes in the
concentrations of photosynthetic pigments, photosystem II
efficiency was slightly higher in the drought-stressed plants
(Fig. 5A) than in the controls. Non-photochemical and
photochemical quenchings (Fig. 5B, 5C) were lower and
higher, respectively, in the stressed plants compared with the
controls, although the differences among treatments were
only significant at week 19. Rubisco initial and total activities
were affected by drought at weeks 13 and 19 (Table 2). The
activation state of Rubisco remained similar in the control
and drought-treated plants.
MDA, soluble sugars, and inulin concentrations
The leaf MDA concentration (Fig. 6A) increased in re-
sponse to water shortage after 19 and 25 weeks of culture.
The root MDA concentration was similar in the control and
drought-treated plants, except at week 19 when it was
significantly higher in the latter than in the former. Water
stress also drastically increased the leaf total soluble sugar
concentration (Fig. 6B), which culminated at the beginning
of the stress exposure at week 13 and then gradually
decreased until the end of the treatment.
Drought increased the endogenous concentrations of
glucose, fructose, sucrose, galactose, and myo-inositol in
leaves at weeks 13 and 19, while sucrose also accumulated
in response to drought at week 25 (Table 3). Mannose and
raffinose were also detected in our leaf samples but re-
mained unaffected in response to drought (detailed data
not shown). The soluble sugar (glucose, fructose, sucrose,
kestose, and nystose) concentrations were not affected by
water stress in the roots (detailed data not shown).
Root inulin concentration, which was quantified at the
different harvests (Table 4), remained unaffected when
expressed on a DW basis. This result, however, implies that
the inulin concentration expressed on a FW basis was
higher in the stressed plants than in the controls at the end
of the treatment period (Table 4). Considering the pre-
viously mentioned root growth inhibition, the total amount
of inulin produced per plant was 54.3 and 28.5 g plant?1for
control and stressed plants, respectively: assuming a plant
density of 150 000 plant ha?1in field conditions, such water
stress would imply a loss of inulin yield corresponding to
3.8 T ha?1. The mean DP remained unaffected in the water-
stressed plants compared with the controls (Table 4).
Because the columns could be considered to be closed
systems and because the volume of solution supplied to
each column was registered during the time course of the
experiment, the water-use efficiency could be estimated. The
synthesis of 1 g of root dry matter required 405662 ml of
solution in control plants and only 244632 ml in stressed
Water stress reduces root growth and inulin yield | 5 of 15
by guest on May 28, 2012
Fig. 2. Evolution of the duration required for the macroscopic appearance of a new leaf (A), total leaf surface (B), leaf mean area (C),
leaf abscission and senescence (D), root FW (E), and shoot-to-root ratio (F) in C. intybus plants exposed to well-watered (control) and
drought (drought) conditions. Water stress was imposed 12 weeks after sowing (grey dashed line), and plants were harvested for
analysis 7, 13, 19, and 25 weeks after sowing. Non-destructive measurements for the duration required for the macroscopic
appearance of a new leaf were made 7, 9, 11, 13, 16, 19, 22, and 25 weeks after sowing. Filled symbols indicate control plants, and
open symbols indicate drought-stressed plants. Each value represents the mean of three replicates, and vertical bars represent the
standard errors. The asterisks indicate the presence of a significant difference between the treatments (*P < 0.05, **P < 0.01,
***P < 0.001).
6 of 15 | Vandoorne et al.
by guest on May 28, 2012
ones. Similarly, the synthesis of 1 g of inulin required
551630 ml of nutrient solution in controls for only 354662
ml in stressed plants.
Water stress had no impact on the protein concentration,
whatever the considered duration of stress (detailed data not
shown). Water stress increased leaf SPS activity (Fig. 7A). In
contrast, water stress had no significant impact on leaf SuSy
activity (Fig. 7B). The acid invertase activity was higher in
stressed leaves than in controls (Fig. 7C) at week 13. This
enzyme activity exhibited an obvious burst at week 25,
although no difference was recorded between the control
and stressed plants at this time. The only difference between
treatments for leaf neutral invertase was recorded at the
end of the experiment, when the drought-stressed plants
exhibited a lower activity than the controls (Fig. 7D). SPS,
SuSy, and invertase activities were also measured in the
roots: the activities of these enzymes decreased with root age,
but water stress had no impact on these activities compared
with controls (detailed data not shown).
The activities of inulin-metabolizing enzymes were quan-
tified in the roots (Table 5). A high 1-SST activity was
recorded by week 7; it progressively decreased with plant
age and to a higher extent in water-stressed plants than in
controls. Such a development-induced decrease was not
recorded for 1-FFT activity, which remained lower in the
drought-stressed plants than in the controls at weeks 19 and
25. An opposite trend was observed for 1-FEH activity,
which was higher at weeks 19 and 25 in the water-stressed
plants than in the controls.
Drought tolerance is a major concern in agronomy and
plant research. In numerous studies performed under
laboratory conditions, drought tolerance is scored based on
an improvement in the survival rate under lethal conditions
that are not necessarily relevant to field conditions. Growth
Fig. 3. Evolution of leaf and root water content (A), leaf water potential (B), corrected leaf osmotic potential (C), and corrected root
osmotic potential (D) in C. intybus plants exposed to well-watered (control) and drought (drought) conditions. The osmotic potentials
were adjusted to the water content of the control plants. All other details are as for Fig. 2.
Water stress reduces root growth and inulin yield | 7 of 15
by guest on May 28, 2012
inhibition may help the plant save and redistribute resour-
ces that become limited under stress, but it can also be
considered counter-productive in terms of yield in agricul-
ture (Skirycz and Inze ´, 2010; Muller et al., 2011; Skirycz
et al., 2011). Carbohydrates generated by photosynthesis
are major building blocks and energy sources for biomass
production and maintenance. Thus, in cultivated plants, de-
termining whether growth inhibition is a consequence of a
stress-induced decrease in photosynthesis appears important.
As far as root chicory is concerned, the present work
clearly showed that growth was strongly affected by drought
conditions, while photosynthesis exhibited resilience to those
environmental constraints. Growth inhibition occurred in
both the shoots and roots, although the former was more
affected than the latter after 19 and 25 weeks of culture.
Drought reduced the number of leaves and the mean leaf
area. The proliferation and subsequent expansion of founder
cells recruited from the shoot apical meristem to form new
leaves may have thus been altered in response to water stress
(Beemster et al., 2005).
The net leaf photosynthesis in root chicory is poorly
influenced by the water regime when expressed on a surface
Table 1. Evolution of the stomatal conductance (gs) and gas
exchange (E and A) in plants of C. intybus exposed to well-
watered (control) and drought (drought) conditions. Water stress
was imposed 12 weeks after sowing, and measurements were
made 11, 13, 16, 19, 22, and 25 weeks after sowing. Each value
represents the mean of five (gs) or 10 (E and A) replicates and is
given with its standard error. Asterisks indicate the presence of
a significant difference between the treatments (*P < 0.05,
**P < 0.01, ***P < 0.001).
Control DroughtControl Drought Control Drought
Fig. 4. Evolution of chlorophyll a (A), chlorophyll b (B), the Chl a/Chl b ratio (C), and carotenoid concentration (D) in C. intybus plants
exposed to well-watered (control) and drought (drought) conditions. All other details are as for Fig. 2.
8 of 15 | Vandoorne et al.
by guest on May 28, 2012
basis, although a decrease in the specific leaf area may
contribute to the decreased photosynthesis on a DW basis.
Stomatal conductance exhibited a weak correlation with net
photosynthesis, as also demonstrated by Monti et al. (2005).
According to these authors, the impact of water stress
on root chicory photosynthesis was mainly due to non-
stomatal causes. Our work demonstrates, for the first time
in this species, that the efficiency of the light phase was
maintained or even increased in stressed tissues, as indicated
by a stress-induced decrease in NPQ and an increase in qP
and uPSII(Fig. 5). A similar observation was reported in the
sunflower (Cechin et al., 2008), but the underlying physio-
logical reasons remain poorly documented. Several osmo-
protectants are known to protect photophosphorylation
and electron transport of chloroplast membranes against
desiccation, particularly for galactinol, which is produced
from galactose and myo-inositol (Nishizawa et al., 2008).
These two compounds were increased in our stressed leaves
Fig. 5. Evolution of uPSII(A), NPQ (B), and qp(C) in C. intybus plants exposed to well-watered (control) and drought (drought) conditions.
Each value represents the mean of five replicates and vertical bars represent the standard errors. All other details are as for Fig. 2.
Table 2. Evolution of the initial and total Rubisco activities (nmol mg prot?1min?1) and activation state (%) in plants of C. intybus
exposed to well-watered (control) and drought (drought) conditions. Water stress was imposed 12 weeks after sowing, and plants were
harvested for analysis 7, 13, 19, and 25 weeks after sowing. Each value represents the mean of six replicates and is given with its
standard error. Asterisks indicate the presence of a significant difference between the treatments (*P < 0.05, **P < 0.01, ***P < 0.001).
Week Initial activity (nmol mg prot?1min?1) Total activity (nmol mg prot?1min?1) Activation state (%)
ControlDrought Control Drought ControlDrought
Water stress reduces root growth and inulin yield | 9 of 15
by guest on May 28, 2012
(Table 3). Both the initial and total activities of Rubisco and
pigment concentrations were reduced in response to water
stress. Such decreases, however, only had a marginal impact
on net photosynthesis, suggesting that both Rubisco and
chlorophylls are present in excess in the control leaves. Wang
et al. (2008) also reported that in addition to a decrease in the
total chlorophyll content, the maintenance of the Chl a/Chl
b ratio is an important parameter involved in the stability of
the light phase of photosynthesis under stress. The response
of Rubisco to water stress varies greatly among plant species
(Parry et al., 2002). The similarity of the activation states in
the control and stressed plants suggests that water stress did
not reduce the in vivo activity of Rubisco, expressed as
a percentage of the carboxylation potential. Nevertheless, this
carboxylation potential was clearly reduced by drought
(Table 2), which could be related to the binding of inhibitors
within the catalytic site of the enzyme.
Plant growth inhibition associated with the maintenance
of photosynthesis activity leads to the accumulation of
soluble sugars within plant cells. This accumulation may
have numerous impacts on plant metabolism because sugars
play key roles in plants, not only as nutrients but also as
signalling and protecting molecules (Bolouri-Moghaddam
et al., 2010; Pagter et al., 2011). The total soluble sugars in
the leaves peaked 1 week after the onset of stress and then
decreased progressively until week 25. The sugar levels,
however, remained higher in the stressed plants than in the
controls. The decrease in sugar concentration recorded in
the control plants is a developmentally regulated process
corresponding to a general decrease in the physiological
activity that occurs at the end of the first year of growth in
this biannual plant species, the time at which the plants
start to cope with the late autumn/winter period. Although
sugar accumulation in stressed plants correlated with the
decrease in osmotic potential, a rough quantitative analysis
based on the mean water content and the use of the Van’t
Hoff equation suggests that the soluble sugars did not con-
tribute more than 8% to the total lowering of the osmotic
Fig. 6. Evolution of root and shoot MDA concentration (A) and leaf total soluble sugars (B) in C. intybus plants exposed to well-watered
(control) and drought (drought) conditions. All other details are as for Fig. 2.
Table 3. Evolution of the free glucose, fructose, sucrose, galactose, and myo-inositol concentrations in the leaves of C. intybus exposed
to well-watered (control) and drought (drought) conditions. Water stress was imposed 12 weeks after sowing, and plants were harvested
for analysis 7, 13, 19, and 25 weeks after sowing. Each value represents the mean of three replicates and is given with its standard error.
Asterisks indicate the presence of a significant difference between the treatments (*P < 0.05, **P < 0.01, ***P < 0.001).
Week Glucose [mmol (g DW)?1] Fructose [mmol (g DW)?1] Sucrose [mmol (g DW)?1]
ControlDrought ControlDroughtControl Drought
Galactose [mmol (g DW)?1]
Myo-inositol [lmol (g DW)?1]
10 of 15 | Vandoorne et al.
by guest on May 28, 2012
Table 4. Evolution of the inulin content expressed on a DW and FW basis, mean DP of the inulin chains, and quantity of inulin in the
roots of C. intybus exposed to well-watered (control) and drought (drought) conditions. Water stress was imposed 12 weeks after
sowing, and plants were harvested for analysis 7, 13, 19, and 25 weeks after sowing. Each value represents the mean of three replicates
and is given with its standard error. Asterisks indicate the presence of a significant difference between the treatments (*P < 0.05, **P <
0.01, ***P < 0.001).
Week Inulin content (g inulin 100 g DW?1)Mean DP Quantity of inulin in roots
Control DroughtControl DroughtControl Drought
Inulin content (g inulin 100 g FW?1)
Fig. 7. Evolution of leaf SPS (A), SuSy (B), acid invertase (C), and neutral invertase (D) activities in C. intybus plants exposed to well-
watered (control) and drought (drought) conditions. All other details are as for Fig. 2.
Water stress reduces root growth and inulin yield | 11 of 15
by guest on May 28, 2012
potential. Other compounds, such as organic acids and
proline, that may assume key roles in osmotic adjustment
(Hummel et al., 2010), were not quantified in the present
The accumulation of the non-reducing sucrose did not
only result from a passive growth inhibition but also from
an active stimulation of SPS activity (Fig. 6). Moreover, the
fact that inulin concentration was not reduced by water
stress (see below) indicates that sucrose transport from
source leaves to sink roots was not severely hampered under
stress. Not only sucrose but also glucose and fructose accu-
mulated at weeks 13 and 19 in the leaves, at a time when the
activities of acid invertase and, to a lesser extent, SuSy
increased in the stressed leaves compared with the control
leaves. Hexokinase is a glucose sensor with separate cata-
lytic and signalling activities (Moore et al., 2003), and the
produced glucose may act as a signalling molecule (Rolland
et al., 2006). Hummel et al. (2010) recently reported that in
Arabidopsis thaliana exposed to water deficit the rosette
relative expansion rate was decreased more than photosyn-
thesis, leading to a more positive carbon balance, and that
water deficit has a limited impact, often stimulating enzyme
activities. In that study, however, root growth was pro-
moted, while it was clearly inhibited in C. intybus. Such a
discrepancy may be related to the pattern of root develop-
ment, which involves organ enlargement after initiation of
the tap root, a process that is especially sensitive to water
shortages but does not occur in A. thaliana. The absence of
a drought effect on the root sugar content confirms the view
of Hummel et al. (2010) that there is no global down-
regulation of carbon metabolism under conditions of soil
water deficit, in contrast to data obtained from drastic
drought protocols involving drying out of the plant or
immersion in osmotica (Verslues et al., 2006).
Water stress hastened the leaf senescence process, at least
between weeks 7 and 19 (Fig. 2). Photosynthesis can be
argued to have remained active in only a small proportion
of the foliage, which, in turn, may influence the global plant
carbon budget. Stress-induced ethylene oversynthesis is fre-
quently reported to hasten senescence (Lim and Nam, 2005)
but may also influence sugar metabolism and leaf devel-
opment (Kendrick and Chang, 2008; Cho and Yoo, 2009).
A slight burst in ethylene synthesis has been noticed in
drought-treated root chicory (S. Lutts, unpublished results).
In this species, however, stress-induced senescence mainly
involves the basal part of the rosette and not the most
photosynthetically active young leaves. Although the ratio
of the total leaf area/root DW decreased in response to the
soil water deficit, a simultaneous decrease in specific leaf
area mitigated the impact of stress on the shoot/root ratio.
Despite senescence processes, modification of the leaf struc-
ture associated with a more efficient photosynthesis light
phase is suggested to allow the plant to maintain sugar
synthesis to fulfil requirements for the accumulation of
reserves in the roots.
Oxidative stress may also be responsible for hastening
senescence. Small soluble sugars and the enzymes associated
with their metabolic pathways are connected to oxidative
stress and reactive oxygen species signalling. Although excess
sugar production in source leaves may result in the gene-
ration of excess cytosolic H2O2, endogenous sugar availabil-
ity may also feed the oxidative pentose phosphate pathway,
creating reducing power for glutathione production (Bolouri-
Moghaddam et al., 2010). In the present case, the highest
MDA concentration was observed at weeks 19 and 25
(Fig. 6A), at a time when soluble sugars had already
decreased in the stressed leaves. In contrast, a low level of
MDA was recorded at week 13, when the total soluble sugar
concentration was the highest, suggesting that sugars may be
involved in quenching reactive oxygen species (Coue ´e et al.,
2006). Similarly, a low level of MDA in the root of C. intybus
could be, at least partly, related to the presence of inulin
because fructans are efficient in membrane protection against
drought and low temperatures (Van Laere and Van den
Ende, 2002; Valluru and Van den Ende, 2008).
According to Schittenhelm (1999), water stress is a major
factor affecting yield in root chicory. The present work shows
that root growth, but not inulin synthesis, was the main
parameter involved in the deleterious impact of the water
shortage. Under our experimental conditions, inulin concen-
tration remained similar in the control and stressed plants.
Although the in vitro activities of the enzymes involved in
inulin synthesis (1-SST and 1-FFT) were significantly reduced
in stressed plants at weeks 19 and 25 (Table 5), this change
had no impact on the final inulin concentration (Table 4).
1-SST is involved in the initiation of inulin chain
synthesis, and its activity regularly decreased from week 7
to the end of the culture. Such a decrease was already
Table 5. Evolution of the 1-SST, 1-FFT, and 1-FEH activities in the roots of C. intybus exposed to well-watered (control) and drought
(drought) conditions. Water stress was imposed 12 weeks after sowing, and plants were harvested for analysis 7, 13, 19, and 25 weeks
after sowing. Each value represents the mean of three replicates and is given with its standard error. Asterisks indicate the presence of
a significant difference between the treatments (*P < 0.05, **P < 0.01, ***P < 0.001).
Week 1-SST (nmol mg prot?1min?1)1-FFT (nmol mg prot?1min?1)1-FEH (nmol mg prot?1min?1)
12 of 15 | Vandoorne et al.
by guest on May 28, 2012
mentioned by De Roover et al. (2000) and corresponds to
a precise pattern of root development. According to Druart
et al. (2001), 1-SST is initiated only after the first phase of
structural root growth, which ends 42 days after seeding.
The corresponding enzyme activity, which is involved in
sink strength regulation (Ame ´ziane et al., 1995), culminated
9 weeks after seeding. It thereafter decreased progressively
until the end of the culture. Water stress effects on 1-SST
may have started at a time when the enzyme activities were
already decreased and almost all of the inulin chains were
initiated. De Roover et al. (2000) noticed that, at the
seedling stage, the drought-induced stimulation of 1-SST
could lead to a short-term accumulation of fructan in the
roots, as recorded 2 weeks after an abrupt stress imposition.
The present work shows, however, that a progressive de-
cline in water availability maintained on a long-term basis
had no detrimental impact on inulin concentration expressed
on a DW basis. This observation suggested that stress-
induced stimulation at the seedling stage should be regarded
Under our experimental conditions, water stress signifi-
cantly reduced 1-FFT activity, which is involved in inulin
chain elongation. The drought conditions also slightly
increased 1-FEH activity, which catalyses inulin chain depoly-
merization. Although a decrease in the mean DP of the
inulin chain was expected, water stress had no significant
impact on this parameter (Table 4). The recorded FEH
activities in the present study (Table 5) remained rather low
compared with a previously reported cold-induced FEH
increase (Van Laere and Van den Ende, 2002). Thus, FEH
likely does not significantly contribute to depolymerization
in vivo. Soluble sugars, particularly sucrose, fructose, and glu-
cose, are frequently reported in the literature as being among
the main parameters influencing the regulation of the
activities of inulin-metabolizing enzymes (Van Laere and
Van den Ende, 2002; Van den Ende et al., 2004, and
references in these studies). The current work was neverthe-
less unable to detect any significant impact of water stress on
the concentration of these compounds within the roots.
Above a certain inulin concentration, a phase transition to
a gel-like state can be hypothesized to occur in the vacuole,
which may sequester and inactivate fructan enzymes in planta
but not in vitro.
In addition to post-translational regulation, transcriptional
regulation has also been reported for inulin-metabolizing
enzymes. Kusch et al. (2009) recently demonstrated that num-
erous biochemical factors (including Ca2+signalling, protein
kinases, and phosphatases) are directly involved in the
modulation of the expression of the gene encoding 1-FFT.
Similarly, the FEH enzyme activity depends on three distinct
isoforms (1-FEHI, IIa, and IIb) that may exhibit various
patterns of regulation (Van Laere and Van den Ende, 2002;
Van den Ende et al., 2004; Le Roy et al., 2007; Kusch et al.,
In conclusion, C. intybus displays a good level of water
stress resistance, which could be at least partly due to its
Mediterranean origin. Plant growth inhibition could be
involved in this resistance strategy but leads to a significant
decrease in the total inulin production. Nevertheless, the
effect of water stress on Rubisco and sugar-metabolizing
activities did not lead to modifications of the inulin
concentration expressed on a DW basis. Further experi-
ments are therefore required to identify the major causes
of root growth inhibition in this species.
This work was supported by the Re ´gion Wallonne (DGTRE)
of Belgium through the subventions D31-1062, D31-1123,
and D31-1175 and by the Fonds National de la Recherche
Scientifique (FNRS; convention no. 18.104.22.168F). The
authors are also very grateful to FNRS for the PhD
research grant of BV (Aspirant FNRS) and to B. Van Pee,
B. Capelle, and H. Dailly (CARI-UCL) for their valuable
Ame ´ziane R, Limani MA, Noctor G, Morot-Gaudry JF. 1995.
Effect of nitrate concentration during growth and carbon partitioning
and sink strength in chicory. Journal of Experimental Botany 46,
Baert JRA. 1997. The effect of sowing and harvest date and cultivar
on inulin yield and composition of chicory (Cichorium intybus L.) roots.
Industrial Crops and Products 6, 195–199.
Beemster GTS, De Veylder L, Vercruysse S, West G,
Rombaut D, Van Hummelen P, Galichet A, Griuissem W, Inze ´ D,
Vuylsteke M. 2005. Genome-wide analysis of gene expression
profiles associated with cell cycle transition in growing organs of
Arabidopsis. Plant Physiology 138, 734–743.
Bolouri-Moghaddam MR, Le Roy K, Rolland F, Van den Ende W.
2010. Sugar signalling and antioxidant network connectons in plant
cells. FEBS Journal 277, 202–207.
Bradford MM. 1976. A rapid and sensitive method for determining
microgram quantities of protein using the principle of pr(otein-dye
binding. Analytical Biochemistry 72, 248–254.
Cechin I, Corniani N, Fumis TD, Cataneo AC. 2008. Ultraviolet-B
and water stress effects on growth, gas echange and oxidative stress
in sunflower plants. Radiation and Environmental Biophysics 47,
Cho YH, Yoo SD. 2009. Emerging complexity of ethylene signal
transduction. Journal of Plant Biology 52, 283–288.
Coue ´e I, Sulmon C, Gouesbet G, El Amrani A. 2006. Involvement
of soluble sugars in reactive oxygen species balance and responses
to oxidative stress in plants. Journal of Experimental Botany 57,
De Roover J, Vandenbranden K, Van Laere A, Van den Ende W.
2000. Drought induces fructan synthesis and 1-SST (sucrose:sucrose
fructosyltransferase) in roots and leaves of chicory seedlings
(Cichorium intybus L.). Planta 210, 808–814.
Devacht S, Lootens P, Roldan-Ruiz I, Carlier L, Baert J,
Van Waes J, Van Bockstaele E. 2009. Influence of low
temperatures on the growth and photosynthetic activity of
Water stress reduces root growth and inulin yield | 13 of 15
by guest on May 28, 2012
industrial chicory, Cichorium intybus L. Photosynthetica 47,
Dielen V, Notte ´ C, Lutts S, Debavelaere V, Van Herck JC,
Kinet JM. 2005. Bolting control by low temperatures in root
chicory (Cichorium intybus var. sativum). Field Crops Research 94,
Druart N, De Roover J, Van den Ende W, Goupil P, Van Laere A,
Rambour S. 2001. Sucrose assimilation during early developmental
stages of chicory (Cichorium intybus L.) plants. Planta 212, 436–443.
Du YC, Nose A, Kawamitsu Y, Murayama S, Wasano K,
Uchida Y. 1996. An improved spectrophotometric determination of
the activity of Ribulose 1,5-bisphopsphate carboxylase. Japanese
Journal of Crop Science 65, 714–721.
Fu JM, Huang BR, Fry J. 2010. Osmotic potential, sucrose level, and
activity of sucrose metabolic enzymes in tall fescue in response to
deficit irrigation. Journal of the American Society for Horticultural
Science 135, 506–510.
Heath RL, Packer L. 1968. Photoperoxidation in isolated
chloroplasts. I. Kinetics and stoeichiometry of fatty acid peroxidation.
Archives of Biochemistry and Biophysics 125, 185–188.
Hendry G. 1993. Evolutionary origins and natural functions of
fructans. A climatological, biogeopgraphic and mechanistic appraisal.
New Phytologist 123, 3–14.
Huber JL, Hite DRC, Outlaw WH, Huber SC. 1991. Inactivation of
highly activated spinach leaf sucrose phosphate synthase by
dephosphorylation. Plant Physiology 95, 291–297.
Hummel I, Pantin F, Sulpice R, Piques M, Rolland G, Dauzat M,
Christophe Pervennt M, Bouteille ´ M, Stitt M, Gibon Y, Muller B.
2010. Arabidopsis plants acclimate to water deficit at low cost through
changes of carbon usage: an integrated perspective using growth,
metabolite, enzyme, and gene expression analysis. Plant Physiology
IPCC. 2007. Fourth assessment report, synthesis report. Contribution
of working groups I, II and III to the fourth assessment report of the
Intergovernmental Panel on Climate Change, core writing team. R. K.
Pachauri, A. Reisinger, eds. IPCC, Geneva.
Kendrick MD, Chang C. 2008. Ethylene signalling: new levels of
complexity and regulation. Current Opinion in Plant Biology 11,
Kerepesi I, Galiba G, Vanilla E. 1998. Osmotic and salt stresses
induced differential alteration in water-soluble carbohydrate content in
wheat seedlings. Journal of Agriculture and Food Chemistry 46,
King SP, Lunn JE, Furbank RT. 1997. Carbohydrate content and
enzyme metabolism in developing canola siliques. Plant Physiology
Kusch U, Greiner S, Steininger H, Meyer AD, Corbvie `re-
Divialle H, Harms K, Rausch T. 2009. Dissecting the regulation of
fructan metabolism in chicory (Cichorium intybus) hairy roots. New
Phytologist 184, 127–140.
Lefe `vre I, Marchal G, Meerts P, Corre ´al E, Lutts S. 2009. Chloride
salinity reduces cadmium accumulation by the Mediterranean
halophyte species Atriplex halimus L. Environmental and Experimental
Botany 65, 145–152.
Le Roy K, Verhaest M, Rabilns A, Clerens S, Van Laere A, Van
den Ende W. 2007. N-glycosylation affects substrate specificity of
chicory fructan 1-exohydrolase: evidence for the presence of an inulin
binding cleft. New Phytologist 176, 317–324.
Lichtenthaler HK. 1987. Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Methods in Enzymology 148,
Lim PO, Nam HG. 2005. The molecular and genetic control of leaf
senescence and longevity in Arabidopsis. Current Topics in
Developmental Bology 67, 49–83.
Marschall M, Proctor MCF, Smirnoff N. 1998. Carbohydrate
composition and invertase activity of the leafy liverwort Porella
platyphylla. New Phytologist 138, 343–353.
Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence – a practical
guide. Journal of Experimental Botany 51, 659–668.
Mingeot D, Dauchot N, Van Cutsem P, Watillon B. 2009.
Characterisation of two cold induced dehydrin genes from Cichorium
intybus L. Molecular Biology Reports 36, 1995–2001.
Monti A, Amaducci MT, Pritoni G, Venturi G. 2005. Growth,
fructan yield, and quality of chicory (Cichorium intybus L.) as related to
photosynthetic capacity, harvest time and water regime. Journal of
Experimental Botany 56, 1389–1395.
Moore B, Zhou L, Rolland F, Hall Q, Cheng WH, Liu YX,
Hwang I, Jones T, Sheen J. 2003. Role of the Arabidopsis glucose
sensor HXK1 in nutrient, light, and hormonal signaling. Science 300,
Muller B, Pantin F, Ge ´nard M, Turc O, Freixes S, Piques M,
Gibon Y. 2011. Water deficits uncouple growth from photosynthesis,
increase C content, and modify the relationships between C and
growth in sink organs. Journal of Experimental Botany 62,
Nishizawa A, Yukinori Y, Shigeoka S. 2008. Galactinol and
raffinose as a novel function to protect plants from oxidative damage.
Plant Physiology 147, 1251–1263.
Pagter M, Lefe `vre I, Arora R, Hausman JF. 2011. Quantitative and
qualitative changes in carbohydrates associated with spring
deacclimation in contrasting Hydrangea species. Environmental and
Experimental Botany 72, 358–367.
Parry MA, Andralojc PJ, Khan S, Lea PJ, Keys AJ. 2002. Rubisco
activity: effects of drought stress. Annals of Botany 89, 833–839.
Pilon-Smits EAH, Ebskamp MJM, Jeuken MJW, Weisbeek PJ,
Smeekens SCM. 1995. Improved performance of transgenic
fructan-accumulating tobacco under drought stress. Plant Physiology
Roberfroid MB, Delzenne NM. 1998. Dietary fructans. Annual
Review of Nutrition 18, 117–143.
Rolland F, Baena-Gonzalez E, Sheen J. 2006. Sugar sensing and
signaling in plants: conserved and novel mechanisms. Annual Review
of Plant Biology 57, 675–709.
Schittenhelm S. 1999. Agronomic performance of root chicory,
Jerusalem artichoke and sugarbeet in stress and non-stress
environment. Crop Science 39, 1815–1823.
Skirycz A, Inze ´ D. 2010. More from less: plant growth under limited
water. Current Opinion in Biotechnology 21, 197–203.
14 of 15 | Vandoorne et al.
by guest on May 28, 2012
Skirycz A, Vandenbroucke K, Clauw P, Maleux K, De Meyer B, Download full-text
Dhondt S, Pucci A, Gonzalez N, Hoeberichts F, Tognetti VB,
Galbiati M, Tonelli C, Van Breusegem F, Vuylsteke M, Inze ´ D.
2011. Survival and growth of Arabidopsis plants given limited water
are not equal. Nature Biotechnology 29, 212–214.
Spollen WG, Nelson CJ. 1994. Response of fructan to water deficit
in growing leaves of tall fescue. Plant Physiology 106, 329–336.
Stoyanova S, Geuns J, Hideg E, Van den Ende W. 2011. The food
additives inulin and stevioside counteract oxidative stress. International
Journal of Food Sciences and Nutrition 62, 207–214.
Valluru R, Van den Ende W. 2008. Plant fructans in stress
environments: emerging concepts and future prospects. Journal of
Experimental Botany 59, 2905–2916.
Van den Ende W, Mintiens A, Speleers H, Amabilis AO, Van
Laere A. 1996. The metabolsim of fructans in roots of Cichorium
intybus during growth, storage and forcing. New Phytologist 132,
Van den Ende W, De Coninck B, Van Laere A. 2004. Plant fructan
exohydrolases: a role in signaling and defense? Trends in Plant
Science 9, 1360–1365.
Van Laere A, Van den Ende W. 2002. Inulin metabolism in dicots:
chicory as a model system. Plant, Cell and Environment 25, 803–813.
Verhaest M, Lammens W, Le Roy K, De Ranter C, Van Laere A,
Rabijns A, Van den Ende W. 2007. Insights into the fine architecture
of the active site of chicoty fructan 1-exohydrolase: 1-kestose as
substrate vc sucrose as inhibitor. New Phytologist 174, 90–100.
Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK.
2006. Methods and concepts in quantifying resistance to drought, salt
and freezing, abiotic stresses that affect plant water status. Plant
Journal 45, 523–539.
Volaire F, Lelie `vre F. 1997. Production, persistence, and water-
soluble carbohydrate accumulation in 21 contrasting populations of
Dactylis glomerata L. subjected to severe drought in the south of
France. Australian Journal of Agricultural Research 48, 933–944.
Wang B, Li Z, Eneji AE, Tian X, Zhai Z, Li J, Duan L. 2008. Effects
of coronatine on growth, gas exchange traits, chlorophyll content,
antioxidant enzymes and lipid peroxoidation in maize (Zea mays L.)
seedlings under simulated drought stress. Plant Production Sciences
Wilson RG, Smith JA, Yonts CD. 2004. Chicory root yield and
carbohydrate composition is influenced by cultivar selection, planting
and harvest date. Crop Science 44, 748–752.
Yemm EW, Willis J. 1954. The estimation of carbohydrates in plant
extracts by anthrone. Biochemical Journal 57, 508–514.
Water stress reduces root growth and inulin yield | 15 of 15
by guest on May 28, 2012