Tree Physiology 24, 1303–1311
© 2004 Heron Publishing—Victoria, Canada
Drought-induced changes in flavonoids and other low molecular
weight antioxidants in Cistus clusii grown under Mediterranean field
conditions
IKER HERNÁNDEZ,1 LEONOR ALEGRE1 and SERGI MUNNÉ-BOSCH1,2
1
Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, E-08028 Barcelona, Spain
2
Corresponding author ([email protected])
Received October 24, 2003; accepted April 30, 2004; published online September 1, 2004
Summary Mediterranean plants have evolved a complex antioxidant defense system to cope with summer drought.
Flavonoids, and particularly flavanols and flavonols, are potent
in vitro antioxidants, but their in vivo significance within the
complex network of antioxidant defenses remains unclear, especially in plant responses to stress. To gain insight into the role
of flavonoids in the antioxidant defense system of Cistus clusii
Dunal, we evaluated drought-induced changes in flavonoids in
leaves and compared the response of these compounds with
that of other low molecular weight antioxidants (ascorbic acid,
tocopherols and carotenoids). Among the antioxidant flavonoids analyzed, epigallocatechin gallate was present in the
greatest concentrations (up to about 5 µmol dm –2). Other
flavanols, such as epicatechin and epicatechin gallate, were
found at concentrations below 0.25 and 0.03 µmol dm –2, respectively. Neither of the antioxidant flavonols analyzed,
quercetin and kaempferol, were detected in C. clusii leaves.
Epigallocatechin gallate, ascorbic acid and α-tocopherol concentrations increased to a similar extent (up to 2.8-, 2.6- and
3.3-fold, respectively) in response to drought, but the kinetics
of the drought-induced increases differed. Epigallocatechin
gallate, epicatechin and epicatechin gallate concentrations increased progressively during drought, reaching maximum values after 30 days of stress. Ascorbic acid concentrations
increased twofold after 15 days of drought, and maximum values were attained after 50 days of drought. In contrast,
α-tocopherol concentrations remained constant during the first
30 days of drought, but increased sharply by 3.3-fold after
50 days of drought. The maximum efficiency of photosystem II
photochemistry and the extent of lipid peroxidation remained
constant throughout the drought period, whereas the redox
state of ascorbic acid and α-tocopherol shifted toward their reduced forms in response to drought, indicating that the concerted action of low molecular weight antioxidants may help
prevent oxidative damage in plants.
Keywords: ascorbic acid, carotenoids, flavanols, oxidative
stress, tocopherols.
Introduction
During summer in Mediterranean climates, plants are exposed
to drought stress, a combination of water deficit, high temperatures and high solar radiation (Di Castri 1981). Drought stress
may increase the formation of reactive oxygen species (ROS),
which play a role in intra- and intercellular signaling at low
concentrations but damage several cellular components (e.g.,
lipids, proteins, nucleic acids) when present at high concentrations (Smirnoff 1993, Doke 1997, Dat et al. 2000, Overmyer et
al. 2003). Plants, especially those living in stressful environments, have evolved complex enzymatic and non-enzymatic
antioxidant defense systems to regulate endogenous ROS concentrations throughout their development; however, oxidative
damage may occur when ROS formation and antioxidant defenses become unbalanced (Doke 1997, Dat et al. 2000,
Mahalingam and Fedoroff 2003).
Among low molecular weight antioxidants, the functions of
carotenoids, tocopherols, ascorbic acid and glutathione in
plant responses to stress have been extensively studied (reviewed by Noctor and Foyer 1998, Asada 1999, Smirnoff and
Wheeler 2000, Munné-Bosch and Alegre 2002a, Strzalka et
al. 2003). However, relatively few studies have explored the
role of secondary metabolic pathways in plant responses to
stress. The phenylpropanoid pathway is responsible for the
synthesis of a diverse array of phenolic metabolites such as
flavonoids, tannins, hydroxycinnamate esters and the structural polymer lignin. These compounds are often induced by
stress and serve specific roles in plant protection, e.g., in
pathogen defense or ultraviolet screening or as antioxidants, or
antiherbivory or structural components of the cell wall (Dixon
and Paiva 1995, Grace and Logan 2000). Structure–function
studies of flavonoids have demonstrated that the position of
their hydroxyl groups, double carbon bonds and modifications
like glycosylation, prenylation and methylation determine
their antioxidant properties (Rice-Evans et al. 1997). Among
flavonoids, the flavanols epicatechin gallate and epigallocatechin gallate, and the flavonol quercetin are excellent antioxidants in vitro, displaying antioxidant activities up to 5-fold
higher than α-tocopherol and ascorbic acid (Rice-Evans et al.
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HERNÁNDEZ, ALEGRE AND MUNNÉ-BOSCH
1997). Flavanols and flavonols have received considerable attention in food science and biomedicine because of their
anti-inflammatory, antiallergic, anti-platelet, antiviral and
antitumoral activities, which are caused, at least in part, by
their potent antioxidant properties (Attaguile et al. 2000).
However, the contribution of these compounds to the antioxidant defense system and their relevance in plant responses to
drought are incompletely understood.
The mechanisms that Cistus clusii Dunal has evolved to
withstand drought are of special interest because this sclerophyllous shrub is typical of plants of the Mediterranean climate zone and grows in extreme climatic conditions. Cistus
clusii is found in littoral brushwood growing in calcareous and
dry soils of the Mediterranean coast, but it can also inhabit
sandy and perturbed areas. It is a branched sclerophyllous
shrub with linear hypostomatic leaves, which are dark green
above, white-tomentose beneath, and can curl downward,
thereby reducing exposure to solar radiation. We have previously shown that hydrogen peroxide accumulates at the onset
of drought and that some mechanisms of photo- and antioxidative protection (including tocopherol and zeaxanthin
synthesis) are activated in drought-stressed C. clusii plants
(Munné-Bosch and Alegre 2002b, Munné-Bosch et al. 2003).
To further explore the mechanisms of drought-stress resistance in this species, the present study was undertaken: (1) to
identify antioxidant flavonoids in leaves of C. clusii plants; (2)
to evaluate the response of antioxidant flavonoids to drought;
and (3) to compare drought-induced changes in flavonoids
with those of other low-molecular-weight antioxidants such as
ascorbic acid, α-tocopherol and carotenoids.
Materials and methods
Plant material and drought stress treatment
Seeds of C. clusii were germinated on moist filter paper, and
seedlings were transferred to 0.5-l pots containing a mixture of
soil:peat:perlite (1:1:1, v/v). The potted seedlings were grown
in a greenhouse at a controlled day/night temperature of
24/18 °C. Seedlings were irrigated twice a week, once with
water and once with Hoagland’s solution. After 1 year, plants
were transplanted to the Experimental Fields at the University
of Barcelona (NE Spain) and grown under Mediterranean field
conditions for 1 year before the experiment began. At the beginning of the experiment, plants received 100 mm of water.
Thereafter, irrigation was withheld for 50 days and the plants
were covered with a clear polyvinyl chloride (PVC) sheet
when it rained. Measurements started on April 23, 2001 and
were taken on clear sunny days about once every 2 weeks
throughout the 50-day period of drought.
Environmental conditions were monitored at a weather station located 8 m from the experimental plot. Photosynthetically active photon flux (PPF), air temperature and relative
humidity were measured at 5-min intervals throughout the
day. The PPF was measured with a quantum sensor (Li-Cor,
Lincoln, NE), and air temperature and relative humidity were
measured with a thermohygrometer (Vaisala, Helsinki, Fin-
land). Vapor pressure deficit was calculated from air temperature and relative humidity data according to Nobel (1991).
During the experiment, PPF, air temperature and vapor pressure deficit at midday ranged between 1832 and 1910 µmol
m –2 s –1, 19.7 and 25.2 °C, and 0.84 and 2.27 kPa, respectively
(Table 1).
Plant water status, leaf mass per area ratio (LMA) and concentrations of malondialdehyde (MDA, an indicator of lipid
peroxidation), chlorophylls, total phenolics and low molecular
weight antioxidants (flavonols, flavanols, ascorbic acid, tocopherols and carotenoids) were measured on fully developed
young leaves collected at predawn (1 h before sunrise). For determination of MDA and pigment and antioxidant concentrations, leaves were collected, frozen in liquid nitrogen and
stored at –80 °C until analyzed.
Plant water status and leaf mass per area ratio
Leaves were weighed and leaf area was immediately measured
with a flatbed scanner (GT-5000, Epson, Nagano, Japan) and
an image processing program. The leaves were then
rehydrated for 24 h at 4 °C in darkness and subsequently dried
for 24 h at 80 °C. Relative leaf water content (RWC) was determined as 100(FM – DM)/(TM – DM), where FM is fresh
mass, TM is turgid mass after re-hydrating the leaves, and DM
is the dry mass after oven-drying the leaves. We determined
LMA as DM/leaf area.
Estimation of lipid peroxidation
The extent of lipid peroxidation in leaves was estimated by
measuring the amount of MDA as described by Munné-Bosch
and Alegre (2003). Leaves were extracted four times with
80:20 (v/v) ethanol:water containing 1 ppm butylated hydroxytoluene (BHT) by ultrasonication (Vibra-Cell Ultrasonic
Processor, Vibra-Cell, Sonics & Materials, Danbury, CT). The
extracts were centrifuged, the supernatants pooled, and an
aliquot of appropriately diluted sample was added to an equal
volume of thiobarbituric acid (TBA) solution containing 20%
(w/v) trichloroacetic acid, 0.01% (w/v) BHT and 0.65% (w/v)
TBA. A blank was prepared by replacing the sample with extraction medium, and controls for each sample were prepared
by replacing TBA with 50 mM NaOH. Samples were heated at
95 °C for 25 min and, after cooling, the (TBA)2-MDA adduct
was isocratically separated on a Hypersyl ODS-5 µm column
(250 × 4.6 mm, Teknokroma, St. Cugat, Spain) by using 5 mM
potassium phosphate buffer (pH 7.0) containing 15% aceto-
Table 1. Photosynthetically active photon flux (PPF), air temperature
(Tair) and vapor pressure deficit (VPD) at midday during the drought
period.
Days of drought
PPF (µmol m –2 s –1)
Tair (°C)
VPD (kPa)
0
15
30
50
1837
1832
1889
1910
25.2
19.7
23.0
21.8
2.27
0.84
1.27
1.37
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ANTIOXIDANTS AND DROUGHT STRESS RESISTANCE IN CISTUS CLUSII
nitrile and 0.6% tetrahydrofuran as eluant at a flow rate of
0.9 ml min –1. Absorbance of the (TBA)2-MDA adduct was
measured at 537 nm (Diode array detector 1000S, Applied
Biosystems, Foster City, CA), and MDA was identified by its
characteristic spectrum and by co-elution with the authentic
standard 1,1,3,3-tetraethoxypropane from Sigma (Steinheim,
Germany), which is stoichiometrically converted to MDA during the acid-heating step of the assay.
Chlorophyll fluorescence measurements
Chlorophyll fluorescence in attached leaves was measured at
predawn with a portable fluorimeter mini-PAM (Walz, Effeltrich, Germany) according to Bilger et al. (1995). Maximum
efficiency of photosystem II photochemistry (Fv /Fm ratio),
which gives an estimate of photoinhibitory damage to the
photosynthetic apparatus, was calculated as (Fm – Fo )/Fm,
where Fm and Fo are the maximum and basal fluorescence
yields, respectively, emitted by leaves that have been exposed
to darkness throughout the night.
Determination of photosynthetic pigments
Photosynthetic pigments were extracted and analyzed by high
performance liquid chromatography (HPLC) as described by
Munné-Bosch and Alegre (2003). Briefly, leaves were ground
in liquid nitrogen and extracted four times with ice-cold acetone by ultrasonication (Vibra-Cell Ultrasonic Processor). Extracts were centrifuged and the supernatants combined.
Pigments were separated on a Dupont non-endcapped Zorbax
ODS-5 µm column (250 × 4.6 mm, 20% C, Teknokroma,
St. Cugat, Spain) at 30 °C for 38 min at a flow rate of 1 ml
min –1. The solvents were (A) acetonitrile:methanol (85:15,
v/v) and (B) methanol:ethyl acetate (68:32, v/v). The gradient
used was: 0–14 min 100% A, 14–16 min decreasing to 0% A,
16–28 min 0% A, 28–30 min increasing to 100% A, and
30–38 min 100% A. Detection was carried out at 445 nm (Diode array detector 1000S, Applied Biosystems). Compounds
were identified by their characteristic spectra and by co-elution with chlorophyll and carotenoid standards, which were
obtained from Fluka (Buchs, Switzerland) and Hoffman-La
Roche (Basel, Switzerland).
Determination of total phenolics
Total phenolics were estimated spectrophotometrically according to Lee et al. (1987). Briefly, leaf samples were extracted four times with 50% (v/v) methanol by ultrasonication
(Vibra-Cell Ultrasonic Processor). After centrifugation, supernatants were pooled, and 2 volumes of 2 N Folin and Ciocalteu’s phenolic reagent (Fluka) and 4 volumes of 17% (w/v)
sodium carbonate were added to 1 volume of leaf extract and
the absorbance was measured at 765 nm. Tannic acid (Sigma,
Steinheim, Germany) was used as a standard for calibration
(Schützendübel et al. 2001).
Determination of antioxidant flavonoids
Antioxidant flavonoids (flavanols and flavonols) were determined by HPLC with a modification of the method described
1305
by Revilla and Ryan (2000). Leaf samples were extracted four
times with methanol by ultrasonication (Vibra-Cell Ultrasonic
Processor). After centrifugation, the resulting supernatants
were pooled, evaporated to dryness, and re-suspended in 2 ml
of methanol. Flavonoids were separated on a Hypersyl
ODS-5 µm column (250 × 4.6 mm, Teknokroma) at a flow rate
of 1 ml min –1. The solvents consisted of (A) methanol:water
(2:98 (v/v), the water being adjusted to pH 3 with phosphoric
acid) and (B) acetonitrile. The gradient started with 100% solvent A for 0–5 min decreasing to 90% A, for 5–13 min decreasing to 82% A, for 13–17 min decreasing to 80% A, for
17–22 min decreasing to 70% A, for 22–26 min decreasing to
62% A, for 26–30 min decreasing to 58% A, for 30–34 min
decreasing to 50% A, for 34–39 min decreasing to 30% A, for
39–44 min decreasing to 0% A in solvent B, and then the column was re-equilibrated to the initial conditions before the
next sample was injected. Detection was carried out at 275 (for
flavanols) and 355 nm (for flavonols) with a Diode array detector 2996 (Waters, Milford, MA). Compounds were identified by their characteristic spectra and by co-elution with
standards obtained from Fluka and Sigma.
The presence of epicatechin (EC), epicatechin gallate
(ECG) and epigallocatechin gallate (EGCG) was confirmed
further by liquid chromatography coupled to mass spectrometry (LC 200, Perkin Elmer, Wellesley, MA, coupled to an Applied Biosystems API 3000 MS detection system) by applying
a declustering potential of 60 V. The gradient used was the
same as described previously, except that the flow rate was
0.8 ml min –1 and formic acid was the solvent, not phosphoric
acid, in solvent A. Individual flavonoids were identified by
comparing their mass spectra with those of standards.
Determination of ascorbic acid
The amounts of reduced and oxidized ascorbic acid in leaves
were determined by HPLC as described by Munné-Bosch and
Alegre (2003). Briefly, leaves were extracted four times with
ice-cold extraction buffer (40% (v/v) methanol, 0.75% (w/v)
m-phosphoric acid, 16.7 mM oxalic acid, 0.127 mM diethylenetriaminepentaacetic acid) by ultrasonication (Vibra-Cell
Ultrasonic Processor). After centrifugation, the supernatants
were pooled, and for determination of reduced ascorbic acid,
0.1 ml of the supernatant was transferred to 0.9 ml of the
mobile phase (24.25 mM Na-acetate (pH 4.8, acetic acid));
0.1 mM diethylenetriaminepentaacetic acid; 0.015% (w/v)
m-phosphoric acid; 0.04% (w/v) octylamine; 15% (v/v) methanol). For determination of total ascorbic acid (reduced plus
oxidized), 0.1 ml of the supernatant was incubated for 10 min
at room temperature in darkness with 0.25 ml of 2% (w/v)
dithiothreitol and 0.5 ml of 200 mM NaHCO3. The reaction
was stopped by adding 0.25 ml of 2% (v/v) sulfuric acid and
0.8 ml of the mobile phase. Ascorbic acid was isocratically
separated on a Spherisorb ODS C8 column (Teknokroma) at a
flow rate of 0.8 ml min –1. Detection was carried out at 255 nm
(Applied Biosystems Diode array detector 1000S). Ascorbic
acid was identified by its characteristic spectrum and by coelution with an authentic standard from Sigma.
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HERNÁNDEZ, ALEGRE AND MUNNÉ-BOSCH
Determination of tocopherols
α-Tocopherol and its oxidation product, α-tocopherol quinone, were determined by HPLC as described by Munné-Bosch
and Alegre (2003). Leaf samples were extracted four times
with ice-cold n-hexane containing 1 ppm BHT by ultrasonication (Vibra-Cell Ultrasonic Processor). After centrifugation, the supernatants were pooled, evaporated to dryness,
and resuspended in 2 ml of methanol. α-Tocopherol and
α-tocopherol quinone were separated on a Partisil 10 ODS-3
column (250 × 4.6 mm, Scharlau, Barcelona, Spain) at a flow
rate of 1 ml min –1. The solvents were (A) methanol:water
(95:5, v/v) and (B) methanol. The gradient used was:
0–10 min 100% A, 10–15 min decreasing to 0% A, 15–20 min
0% A, 20–23 min increasing to 100% A, and 23–28 min 100%
A. α-Tocopherol and α-tocopherol quinone were quantified
by determining absorbance at 283 and 256 nm, respectively
(Applied Biosystems Diode array detector 1000S). Both compounds were identified by their characteristic spectra and by
co-elution with authentic standards provided by Sigma and
Prof. Strzalka (Jagiellonian University, Krakov, Poland).
Results
Drought did not induce oxidative damage
The RWC of C. clusii plants remained constant at about 82%
during the first 15 days of drought and then decreased progressively to about 75 and 63% after 30 and 50 days of drought, respectively. Though RWC remained constant during the first
15 days of drought, LMA increased 1.7-fold during this period
as a result of a 50% decrease in leaf area. Thereafter, LMA remained constant at around 3 g dm –2, while RWC decreased
(Figure 1). No significant changes in chlorophyll (Chl) a + b
concentration, Chl a/b ratio or MDA concentration, an indicator of lipid peroxidation, were observed in the leaves during
the drought period. Also, the Fv /Fm ratio, which is indicative of
photoinhibitory damage to the photosynthetic apparatus, remained constant at about 0.8 throughout the study (Figure 2).
Drought-induced changes in antioxidant flavonoids
Among the antioxidant flavonoids analyzed, the flavanols EC,
ECG and EGCG were found in leaves, whereas the flavonols
quercetin and kaempferol were not detected. The concentrations of EC and ECG were below 0.25 and 0.03 µmol dm –2, respectively. Among the flavanols, EGCG was present in the
highest concentrations, ranging from 1.97 to 4.99 µmol dm –2
(Figure 3), similar to the concentrations of the carotenoids
lutein and β-carotene, but 20-fold lower than the ascorbic acid
concentration, which was the antioxidant found at highest concentrations in C. clusii leaves.
The concentration of EGCG, which began to increase at the
onset of drought, reached a maximum after 30 days of drought
treatment when the RWC was about 75%. As the drought progressed further, the EGCG concentration remained unchanged, even though RWC decreased to about 63% (Figure 3). The EGCG accounted for between 1.8 and 2.6% of total
phenolics and both parameters showed a close positive corre-
Figure 1. Changes in relative leaf water content (RWC), leaf area and
leaf mass per area ratio (LMA) in C. clusii plants exposed to drought
in the field for 50 days. Data correspond to the mean ± SE of six independent measurements made on leaves collected at predawn (1 h before sunrise).
lation (r 2 = 0.92) during the experiment. Concentrations of EC
and ECG were 20- and 100-fold lower, respectively, than the
concentration of EGCG, but they changed in parallel with the
EGCG concentration during the drought treatment (Figure 3).
Drought-induced changes in other low-molecular-weight
antioxidants
Ascorbic acid concentration increased 2.1-fold after 15 days
of drought treatment when RWC was still unchanged and
reached a maximum concentration of about 97 µmol dm –2 after 50 days of drought (Figure 4). Ascorbic acid concentrations
were positively correlated with EGCG concentrations during
the drought treatment (r 2 = 0.75). In contrast, dehydroascorbic
acid decreased significantly after 15 days of drought, and then
remained unchanged for the remainder of the drought period.
As a result, the redox state of ascorbic acid, expressed as
Dha/Asct (where Dha is dehydroascorbic acid and Asct is total
ascorbic acid) shifted toward its reduced form after 15 days of
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ANTIOXIDANTS AND DROUGHT STRESS RESISTANCE IN CISTUS CLUSII
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Figure 2. Changes in chlorophyll (Chl)
a + b concentration, Chl a/b ratio,
maximum efficiency of photosystem II
photochemistry (Fv /Fm) and malondialdehyde (MDA) concentration in
leaves of C. clusii plants exposed to
drought in the field for 50 days. Data
correspond to the mean ± SE of four
independent measurements made on
leaves collected at predawn (1 h before
sunrise).
drought and remained constant at about 0.18 for the remainder
of the drought (Figure 4).
In contrast to flavanols and ascorbic acid, α-tocopherol concentrations were constant during the first 30 days of drought,
but they increased 3.3-fold after 50 days of drought, when
RWC decreased below 70% (Figure 5). α-Tocopherol quinone, the oxidation product of α-tocopherol, was present in low
concentrations (2 nmol dm –2) throughout the experiment, corresponding to an α-tocopherol concentration of about 2%. As
a result of net α-tocopherol synthesis during the drought period, the redox state of α-tocopherol, estimated as α-TQ/α-Tt
(where α-TQ is α-tocopherol quinone and α-Tt is α-tocopherol plus α-TQ) shifted toward its reduced state after
50 days of drought when RWC was below 70%.
Carotenoid concentrations were variable, but tended to increase in response to drought (Figure 6). Lutein and β-carotene concentrations remained unchanged during the first
30 days of drought, and increased by about 50% when RWC
decreased below 70% (Figure 6). Other carotenoids such as
violaxanthin and neoxanthin changed in parallel with lutein,
and the de-epoxidation state of the xanthophyll cycle remained
below 0.1 throughout the experiment (data not shown).
Discussion
Studying Mediterranean plants within their natural habitat
may reveal novel mechanisms of resistance to environmental
stresses. Cistus clusii is an excellent model for studying plant
responses to drought and, more particularly, for evaluating the
relevance of antioxidant flavonoids in stress resistance, because it is an autochthonous shrub that accumulates large
amounts of phenolics in leaves and is well adapted to withstand summer droughts in Mediterranean field conditions.
Previous studies have demonstrated the presence of flavanols in plant species belonging to the genus Cistus (Poestsch
and Reznik 1972, Vogt and Gülz 1991, Demetzos and Perdetzoglou 1999), and we now report the occurrence of the
flavanols EC, ECG and EGCG in C. clusii leaves. Among the
flavanols, EGCG was present in greatest concentration (about
5 µmol dm –2), whereas EC and ECG were found at concentrations below 0.25 and 0.03 µmol dm –2, respectively, which is in
agreement with the concentrations found for other species
(Jeyaramraja et al. 2003, Kirakosyan et al. 2003). We did not
detect the flavonols quercetin and kaempherol in C. clusii
leaves, which is consistent with previous reports (Vogt et al.
1987).
Drought in the field resulted in decreases in RWC of
C. clusii plants, with values reaching about 75 and 63% after
30 and 50 days of drought, respectively. In a previous study,
we used pressure–volume curves to determine that turgor in
C. clusii leaves is lost at an RWC of about 75% (unpublished
results). Thus, in the present study, the drought treatment probably limited water availability to C. clusii leaves after 30 days.
However, this stress did not negatively affect the biochemical
function of leaves, as indicated by constant MDA concentrations and Fv /Fm ratios during the 50-day drought treatment
(Lamont and Lamont 2000).
Flavanols bear two hydroxyl groups at positions 3′ and 4′ of
the B ring, which confer high antioxidant properties (Rice-Evans et al. 1997). Epicatechin is synthesized from cyanidin by
the action of the enzyme anthocyanidin reductase, which is localized in the cytoplasmic face of the endoplasmic reticulum
(Marles et al. 2003). Esterification of EC and epigallocatechin,
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HERNÁNDEZ, ALEGRE AND MUNNÉ-BOSCH
Figure 4. Changes in the concentrations of ascorbic acid (Asc) and its
oxidation product dehydroascorbic acid (Dha) and in the redox state
of ascorbic acid, estimated as Dha/Asct (where Asct = Asc + Dha), in
leaves of C. clusii plants exposed to drought in the field for 50 days.
Data correspond to the mean ± SE of four independent measurements
made on leaves collected at predawn (1 h before sunrise).
Figure 3. Changes in the concentrations of total phenolics, epicatechin (EC), epicatechin gallate (ECG) and epigallocatechin gallate
(EGCG) in leaves of C. clusii plants exposed to drought in the field for
50 days. Data correspond to the mean ± SE of three independent measurements made on leaves collected at predawn (1 h before sunrise).
Abbreviation: nd = not detected.
which is formed by the action of the same enzyme from
delphinidin, with gallic acid gives rise to ECG and EGCG, respectively, which show enhanced antioxidant activity compared with EC (Rice-Evans et al. 1997, see Figure 7). Though
it remains uncertain where these compounds accumulate in
plant cells, they are relatively lipophilic and may therefore be
associated with membranes (Caturla et al. 2003, Marles et al.
2003). In addition, the flavanols EC, ECG and EGCG can
polymerize and accumulate in vacuoles as condensed tannins
(Marles et al. 2003).
The flavanols EC, ECG and EGCG increased significantly
in drought-stressed C. clusii plants. To our knowledge,
drought-induced changes in these flavanols have previously
been reported only in tea plants (Jeyaramraja et al. 2003) and
in two species of the genus Crataegus (Kirakosyan et al.
2003). In both studies, EC concentrations increased in
drought-stressed plants, in agreement with our results. However, ECG and EGCG decreased in drought-stressed tea
plants, which contrasts with what occurs in C. clusii. This difference may reflect the magnitude of the drought stress imposed on the plants: drought was imposed on field-grown
C. clusii plants, whereas drought was imposed on potted
greenhouse-grown tea plants. Further studies under similar
growth conditions are needed to establish differences among
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ANTIOXIDANTS AND DROUGHT STRESS RESISTANCE IN CISTUS CLUSII
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Figure 6. Changes in the concentrations of the carotenoids lutein and
β-carotene in leaves of C. clusii plants exposed to drought in the field
for 50 days. Data correspond to the mean ± SE of four independent
measurements made on leaves collected at predawn (1 h before sunrise).
Figure 5. Changes in the concentrations of α-tocopherol (α-T) and its
oxidation product α-tocopherol quinone (α-TQ) and in the redox state
of α-tocopherol, estimated as α-TQ/α-Tt (where α-Tt = α-T + α-TQ),
in leaves of C. clusii plants exposed to drought in the field for 50 days.
Data correspond to the mean ± SE of four independent measurements
made on leaves collected at predawn (1 h before sunrise).
species in the accumulation of EC gallate esters in response to
drought.
The response of flavanols and other low-molecular-weight
antioxidants in drought-stressed plants may be attributed partially to leaf morphological changes and metabolic alterations
that prevent oxidative damage in leaves. During the first
15 days of drought, RWC remained unaltered, but flavanols
and ascorbic acid per leaf area increased significantly. In contrast, EGCG and ascorbic acid concentrations decreased
slightly (by 7%) or remained constant, respectively, when expressed on a dry mass basis, indicating that changes in EGCG
and ascorbic acid responded to drought-induced morphological alterations of leaves during the first 15 days. Beside a reduction in leaf area because of leaf shrinkage, it is likely that
the reduction in leaf area and concomitant increase in LMA,
were caused by the development of new leaves when RWC
was still above 80%, as reported previously for the same
Figure 7. (A) Chemical structure of the flavanols epicatechin (E),
epicatechin gallate (ECG) and epigallocatechin gallate (EGCG)
found in C. clusii leaves, which in nature depends on the radicals R1
and R2. (B) Chemical structure of gallic acid, which is present in some
flavanols.
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species (Munné-Bosch et al. 2003).
Leaf area and LMA remained unaltered as drought progressed further, which may be associated with the cessation of
growth caused by reductions in leaf turgor. Between Days 15
and 30, EGCG per leaf area increased by about 67%, whereas
leaf area and LMA remained unaltered and RWC decreased to
about 75%, indicating that the changes were caused by enhanced synthesis of flavanols during drought, rather than alterations in leaf morphology. As RWC decreased further
reaching about 63% on Day 50, the EGCG concentration remained unaltered, suggesting that rates of EGCG degradation
and synthesis were similar. It is unlikely that factors other than
drought were responsible for the observed changes, because
environmental conditions, such as PPF and air temperature
changed only slightly during the study period. Any effect of
leaf aging may also be excluded, because the life span of
C. clusii leaves is more than 1 year.
It has been suggested that flavanols function as antioxidants
in plants (Rice-Evans et al. 1997, Grace and Logan 2000).
Epicatechin, ECG and EGCG are efficient chain-breaking antioxidants and transition metal chelators, thus they may help
inhibit lipid peroxidation (Caturla et al. 2003, Potapovich and
Kostyuk 2003). Other functions have also been suggested for
flavanols. When photosynthetic electron transport is limited,
metabolic alterations may lead to drought-induced increases
in flavanols as a result of the activation of alternative metabolic routes mediated by phenylalanine ammonia lyase (PAL)
and other enzymes. This may lead to accumulation of flavanols and derivatives, such as tannins, which may accumulate in
vacuoles and have an anti-herbivore role (Dixon and Paiva
1995). These various roles are not mutually exclusive, and it is
likely that flavanols, like the other antioxidants, serve several
functions in plants.
Ascorbic acid increased in parallel with total phenolics,
showing a strong positive correlation. This may be associated
with a putative interaction between both groups of compounds, because phenoxyl radicals, i.e., the oxidation products
of phenolics, may be recycled back to their reduced forms by
ascorbic acid. Among phenolics, it is likely that flavanols cooperate with ascorbic acid in the scavenging of reactive oxygen species. Although the subcellular distribution of the
flavanols EC, ECG and EGCG in plant cells is unknown, the
last steps in the synthesis of both flavanols and ascorbic acid
occur in the cytoplasm (Smirnoff and Wheeler 2000, Marles et
al. 2003), suggesting that a physical interaction between both
antioxidants at membrane interfaces is possible. The redox
state of ascorbic acid shifted toward its reduced form at the onset of drought and then remained unaltered, indicating that
ascorbic acid is efficiently recycled once oxidized in droughtstressed plants (Noctor and Foyer 1998, Asada 1999). We
therefore hypothesize that flavanols, similar to α-tocopherol
but probably with a different subcellular location, function as
antioxidants in drought-stressed C. clusii plants, and that the
resulting phenoxyl radicals are recycled back to their reduced
forms by ascorbic acid, which in turn, is recycled back to its
reduced form, thereby maintaining its redox state as drought
progresses.
In contrast to EGCG and ascorbic acid, α-tocopherol and
carotenoid concentrations increased in C. clusii leaves only
when the RWC was below 70%. These two groups of antioxidants are known to cooperate in the protection of thylakoids
from oxidative stress, particularly in the scavenging of singlet
oxygen (Havaux 1998, Trebst et al. 2002). α-Tocopherol
quinone, which is the product of singlet oxygen scavenging by
α-tocopherol, remained constant throughout the drought treatment, indicating cooperation between tocopherols and carotenoids in the control of singlet oxygen concentrations in
thylakoids of drought-stressed C. clusii plants. As a result of
increases in α-tocopherol and the maintenance of α-tocopherol quinone concentrations, the redox state of α-tocopherol
shifted toward its reduced state. As suggested for ascorbic
acid, α-tocopherol may participate in functions that are not directly associated with the protection of photosynthetic membranes from oxidative damage. Though it is still to be demonstrated, it is likely that α-tocopherol regulates enzymatic
activity by affecting the fluidity of thylakoids or altering
intracellular signaling by modulating jasmonic acid synthesis,
as suggested previously (Munné-Bosch and Alegre 2002a).
In conclusion, we report (1) the presence of the flavanols
EC, ECG and EGCG in C. clusii leaves; (2) drought-induced
increases in flavanols in field-grown plants; and (3) parallel
changes in flavanols and other low-molecular-weight antioxidants in drought-stressed plants. Increased syntheses of flavanols, ascorbic acid, tocopherols and carotenoids in C. clusii
leaves showed different kinetics in response to drought. To
better understand the antioxidant role of flavanols, further research is needed to characterize their subcellular distribution
and oxidation products.
Acknowledgments
This study was supported by the Ministerio de Ciencia y Tecnología
(projects MCYT BOS2000-0560 and MCYT BOS2003-01032). We
are grateful to Prof. Kazimizierz Strzalka (Jagiellonian University,
Krakov, Poland) for kindly providing α-tocopherol quinone, and the
Serveis Científico-Tècnics and Serveis dels Camps Experimentals
(University of Barcelona) for technical assistance.
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