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Plant Biosystems - An International Journal Dealing
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Physiological and antioxidant responses of Quercus
ilex to drought in two different seasons
a
bc
bc
d
bc
Isabel Nogués , Joan Llusià , Romà Ogaya , Sergi Munné-Bosch , Jordi Sardans , Josep
bc
Peñuelas
a
& Francesco Loreto
e
Consiglio Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale, Italy
b
CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Spain
c
CREAF, E-08193 Cerdanyola del Vallès, Spain
d
Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Spain
e
Consiglio Nazionale delle Ricerche, Istituto per la Protezione delle Piante, Italy
Accepted author version posted online: 23 Jan 2013.Published online: 21 Mar 2013.
To cite this article: Isabel Nogués, Joan Llusià, Romà Ogaya, Sergi Munné-Bosch, Jordi Sardans, Josep Peñuelas & Francesco
Loreto (2014) Physiological and antioxidant responses of Quercus ilex to drought in two different seasons, Plant Biosystems
- An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, 148:2,
268-278, DOI: 10.1080/11263504.2013.768557
To link to this article: http://dx.doi.org/10.1080/11263504.2013.768557
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Plant Biosystems, 2014
Vol. 148, No. 2, 268–278, http://dx.doi.org/10.1080/11263504.2013.768557
Physiological and antioxidant responses of Quercus ilex to drought in
two different seasons
ISABEL NOGUÉS1*, JOAN LLUSIÀ2,3, ROMÀ OGAYA2,3, SERGI MUNNÉ-BOSCH4,
JORDI SARDANS2,3, JOSEP PEÑUELAS2,3, & FRANCESCO LORETO5
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1
Consiglio Nazionale delle Ricerche, Istituto di Biologia Agroambientale e Forestale, Italy; 2CSIC, Global Ecology Unit CREAFCEAB-CSIC-UAB, Spain; 3CREAF, E-08193 Cerdanyola del Vallès, Spain; 4Departament de Biologia Vegetal, Facultat de
Biologia, Universitat de Barcelona, Spain and 5Consiglio Nazionale delle Ricerche, Istituto per la Protezione delle Piante, Italy
Abstract
Climate change projections forecast a warming and an associated change in the distribution and intensity of rainfalls. In the
case of the Mediterranean area, this will be reflected in more frequent and severe drought periods in the future. Within a longterm (9 years) manipulation experiment, we aimed to study the effect of the soil drought projected for the coming decades (an
average of 10% soil moisture reduction) onto photosynthetic rates and water relations, and onto the antioxidant and antistress defense capacity of Quercus ilex, a dominant species in Mediterranean forests, in two different seasons, spring and
summer. Results showed that photosynthesis was limited by stomatal closure in summer. However, a decrease in
photosynthesis as a consequence of drought was observed only during spring, possibly due to a low pigment concentration
and to an insufficient antioxidant protection. In summer, the increased resistance to CO2 entry reduced photosynthesis in
control and drought-treated leaves, though the higher pigment content and antioxidant levels in summer leaves prevented a
further decrease in photosynthesis as a consequence of drought. Also total monoterpene emission rates were higher in
summer than in spring, though they did not change with drought, as happened with photosynthetic pigments. On the other
hand, the antioxidant defense system was induced by drought in both studied seasons, indicating an efficient activation of
defense responses aiming at scavenging reactive oxygen species produced in Q. ilex leaves under drought.
Keywords: Antioxidants, climate change, isoprenoids, photosynthesis, photosynthetic pigments
Abbreviations: A, photosynthesis; gs, stomatal conductance; Fv/Fm, ratio between variable and maximal fluorescence;
RWC, relative leaf water content; ROS, reactive oxygen species; ASC, ascorbate; DHA, dehydroascorbate; GSH, glutathione;
CAT, catalase; APX, ascorbate peroxidase; GR, glutathione reductase; Chl, chlorophyll; VZA, total amount of the xanthophyll
cycle components; DPS, de-epoxidation state of the xanthophyll cycle
Introduction
Mediterranean summer is characterized by low
precipitation, high temperature, and high irradiance.
Moreover, current climate projections predict drier
and warmer conditions for the Mediterranean basin
in future decades (IPCC 2007). Despite being
adapted to the environment, climate change may
further affect the physiological activity of Mediterranean plants. Mediterranean summer drought is
generally considered the primary constraint to the
productivity and distribution of Mediterranean
vegetation (Larcher 2000). Many studies have
described reductions in photochemical efficiency
and low photosynthetic rates during summer drought
(Tenhunen et al. 1990; Peñuelas et al. 1998; Llorens
et al. 2003; Vitale et al. 2012).
Reactive oxygen species (ROS) production
increases in plants under stress conditions such as
high light, drought, low temperature, high temperature, and mechanical stress. ROS can damage
cellular components, but they are involved in growth
regulation, development, responses to environmental
stimuli, and cell death. Thus, the redox equilibrium
and the capacity of ROS scavenging have a key role
for the normal development of a plant and for the
perception, signaling, and adaptation to stress. The
level of ROS in cells is determined by the interplay
Correspondence: I. Nogués, Istituto di Biologia Agroambientale e Forestale, Consiglio Nazionale delle Ricerche, Via Salaria Km. 29 300 – 00015
Monterotondo Scalo, Roma, Italy. Tel: þ39 06 90672530. Fax: þ39 06 9064492. Email: [email protected]
q 2013 Società Botanica Italiana
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Quercus ilex responses to field drought
between ROS-producing pathways and ROS-scavenging mechanisms that include superoxide dismutase,
catalase (CAT), enzymes, and metabolites from the
ascorbate (ASC) – glutathione (GSH) cycle, tocopherols, and carotenoids, including the xanthophylls,
violaxanthin, zeaxanthin and antheraxanthin (VZA)
(Noctor & Foyer 1998; Munné-Bosch 2005).
Volatile isoprenoids also seem to play an antioxidant
role, by quenching ROS and strengthening membranes of plants exposed to high temperatures and
drought (Vickers et al. 2009).
Isoprenoid emission rates and internal concentrations are affected by virtually all environmental
factors, temperature being the most important driver
of the emissions (Peñuelas & Llusia 2001; Loreto &
Schnitzler 2010). The impact of drought is controversial. There are several studies showing
reduction in isoprenoid emission rates under severe
drought stress (Delfine et al. 2005; Peñuelas &
Staudt 2010), possibly as a consequence of reduced
photosynthetic activity and reduced carbon availability. However, isoprenoid inhibition rarely occurs
when drought is mild (Peñuelas & Staudt 2010) and
the emission of isoprene (the most abundant of the
volatile isoprenoids) may even be stimulated when
plants recover from drought (Sharkey & Loreto
1993; Brilli et al. 2007).
Plants have evolved specific acclimation and
adaptation mechanisms to respond to long-term
drought stresses. Analysis of these protective mechanisms will contribute to our knowledge of tolerance
and resistance to stress. The complex responses to
environmental stress, which include biochemical and
physiological changes, need to be considered at a
global level to study the multiple interactive
components in this process. On this basis, the
objective of our study was to investigate the impact of
drought on photosynthesis and the protection
mechanisms of Quercus ilex, a dominant species in
Mediterranean forests. Under climate change pressure, drought episodes are believed to occur more
often, and also during spring or summer, under very
different physiological status of the plants. For this
purpose, a field experiment was conducted that
simulated drought conditions projected by global
circulation and ecophysiologically based models
(IPCC 2007; Sabaté et al. 2002) for the coming
decades (10 – 15% decreased soil moisture), with
measurements being carried out in two different
seasons, spring and summer.
Material and methods
The study site and species description
This study was carried out in a Q. ilex (holm oak)
forest in the Prades Mountains of southern Catalonia
269
(418130 N, 08550 E), on a south-facing slope (25%
slope) at 930 m above sea level. The soil is a stony
xerochrept on bedrock of metamorphic sandstone,
and its depth ranges between 35 and 90 cm. Summer
drought is pronounced and usually lasts for 3
months. This holm oak forest is very dense
(1.66 trees/m2). Trees are about 4 – 8 m high and
have a mean stem diameter (measured at 50 cm
height) of 5 cm. The forest is not monospecific but is
dominated by Q. ilex L., Phillyrea latifolia L., and
Arbutus unedo L. with abundant presence of other
evergreen species well adapted to drought conditions
(Erica arborea L., Juniperus oxycedrus L., and Cistus
albidus L.), and occasional individuals of deciduous
species, such as Sorbus torminalis (L.) Crantz and
Acer monspessulanum L.
Experimental design
Eight 15 £ 10 m plots were marked out at the same
altitude along the slope. Half the plots received the
dry treatment and the other half were used as control
plots. The dry treatment consisted of partial rain
exclusion, achieved by suspending PVC strips at a
height of 0.5 – 0.8 m above the soil. The strips
covered approximately 30% of the total plot surface.
In addition, a 0.8-m-deep ditch was excavated along
the entire top edge of the treatment plots to intercept
run-off water supply. This drought treatment was
started in March 1999 and is still on. Temperature
and precipitation were monitored every half hour by
an automatic meteorological station installed
between the plots in a forest gap. Soil moisture was
measured every 2 weeks throughout the experiment,
as described in Sardans and Penuelas (2007).
Simultaneous samplings and measurements for
this study were carried out during two field
campaigns on April (spring) and July 2008
(summer). Sampling dates (22nd of April and 10th
of July) were chosen as dates with mean temperature
and soil moisture throughout the spring and summer
seasons of the last 10 years (data not shown). Shoot
water potential, chlorophyll (Chl) fluorescence, and
gas exchange were measured in 24 leaves (one leaf
from three different plants per plot) in spring and
summer. In April, leaves were from the previous year;
so, leaves were 1 year old. In July, fully developed
current-year leaves (3 –4 month old) were sampled.
For biochemical assays (determination of ASC,
GSH, total phenolic compounds, antioxidant
enzyme activities, and photosynthetic pigments), 24
samples (three replicates per plot) were taken for
each season. Samples were taken at midday (11.00 –
13.00 h, solar time) the 23rd of April (spring) and the
11th of July (summer), and were frozen immediately
in order to avoid any biological change.
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I. Nogués et al.
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Ecophysiological measurements
The leaf water potential (c) was measured at midday
(11.00 –13.00 h, solar time) in 24 stems (three stems
per plot) using a Scholander-type pressure chamber
(3000 series, soil moisture Equipment Corp., Santa
Barbara, CA, USA).
Leaf net exchange rates of CO2 (photosynthesis,
A) and water (transpiration, E) were measured in situ
with portable gas exchange systems by ADCpro
(ADC BioScientific Ltd Hoddesdon, Herts, UK)
and CID (CI-340 Hand-Held Photosynthesis System, Inc., Camas, WA, USA) which also measured
air temperatures, photosynthetically active radiation
(PAR), and relative humidity in the leaf chamber at
the moment of the leaf gas exchange measurements.
Stomatal conductance (gs) was calculated using the
classic formulation by Von Caemmerer and Farquhar
(1981). Measurements were taken in the morning
(from 30 min after sunrise to 11.30 h, solar time).
Leaves were measured in the original position in the
canopy, under full sunlight.
Chl fluorescence was measured at midday
(11.00 –13.00 h, solar time). Measurements were
carried out with a portable Fluorometer PAM-2000
(Heinz Walz GmbH, Eichenring 6, D-91090
Effeltrich, Germany). After a dark adaptation period
of at least 30 min, the Fv/Fm parameter (the ratio
between variable and maximal fluorescence) was
measured, representing the maximum efficiency of
photosynthetic energy conversion of PSII.
After carrying out gas exchange measurements,
the same leaves were used for collection and
measurement of isoprenoid emission, under the
same environmental conditions (PAR, relative
humidity, and air temperature) as for gas exchange
measurements. The air leaving the gas exchange
cuvette was passed through a Teflon-made T to a
glass tube (8 cm long and with 0.3 cm internal
diameter). The tube was filled manually with the
adsorbents Carbopack B, Carboxen 1003, and
Carbopack Y (Supelco, Bellefonte, PA, USA)
separated by plugs of quartz wool. Prior to use, all
tubes were conditioned for 10 min at 3508C with a
stream of purified helium. A calibrated air sampling
pump was used to standardize air flow through the
absorption tube (Qmax air sampling pump;
Supelco). The sampling time was 5 min, and the
flow varied between 470 and 500 ml min21 depending on the resistance made by the glass tube
adsorbent and quartz wool packing. The trapping
and desorption efficiency of liquid and volatilized
standards such as a-pinene, b-pinene, or limonene
was practically 100%. In order to eliminate the
problem of residual sample in the system, blanks of
5-min air sampling in empty cuvettes were carried
out immediately before each measurement. After
sampling, the glass tubes were stored in a portable
fridge at 48C and taken to the laboratory. There, the
tubes were stored at 2 288C until analysis (within
24 – 48 h). There were no observable changes in
isoprenoid concentrations after storage of the tubes
as checked by analyzing replicate samples immediately and after 48-h storage.
Isoprenoid analyses were carried out by using a
GC-MS system (Hewlett Packard HP59822B, Palo
Alto, CA, USA). Tubes with trapped emitted
isoprenoids were inserted automatically by a robotic
sample processor (FOCUS) (ATAS GL International, Veldhoven, the Netherlands) in an
OPTIC3 injector (ATAS GL International) and
passed into a 30 m £ 0.25 mm £ 0.25 mm film thickness capillary column (Supelco HP-5, Crosslinked
5% Me Silicone, Supelco, Inc., Bellefonte, PE,
USA), desorbed at 2508C for 3 min, and injected into
the same chromatographic column described above.
After sample injection, the initial temperature (408C)
was increased at 308C min21 up to 608C, and
thereafter at 108C min21 up to 1508C maintained
for 3 min, and thereafter at 708C min21 up to 2508C,
which was maintained for another 5 min. Helium
flow was 1 ml min21. The identification of monoterpenes was conducted by GC-MS and compared
with standards from Fluka (Buchs, Switzerland),
literature spectra, and comparison with the library of
the GCD Chemstation G1074A HP. The internal
standard dodecane, which does not mask any
monoterpene, was injected every five sample
analyses. In addition frequent calibration with
a-pinene, 3-carene, b-pinene, b-myrcene, p-cymene, limonene, sabinene, and a-caryophyllene standards was made for standardization of results and
data quantification. Isoprenoid calibration curves
(n ¼ 4 different isoprenoid concentrations) showed
always highly significant (r 2 . 0.98, p , 0.001)
relationships between the peak area of signals and
concentrations. Emission rate calculations were
made on a mass balance basis, after subtracting the
control samples (empty cuvettes) from plant sample
chromatograms.
Biochemical analyses
Total phenolic compounds were extracted twice with
80% methanol (1.5 ml) for 3 min in a ultrasonic bath.
The amount of extracted total phenolic compounds
was determined with the Folin – Ciocalteu reagent
(Singleton & Rossi 1965). Gallic acid was used as
standard and the total phenolic compounds were
expressed as mg of gallic acid equivalents (GAE) per
g of dry matter.
For the determination of reduced ASC and
dehydroascorbate (DHA), the leaf tissue (about
100 mg wet weight) was dissolved in 1.5 ml of 3%
Quercus ilex responses to field drought
determined by consumption of H2O2 (Dhindsa et al.
1981) that was monitored spectrophotometrically
at 240 nm (1 ¼ 0.0435 mM21 cm21). The activity
was expressed as mmol H2O2 min21 mg21 protein.
Protein concentrations were determined spectrophotometrically using Coomassie brilliant blue
R-250 (Bradford 1976). All assays were carried
out at 258C.
25
(a)
Air temperature (˚C)
20
15
10
Foliar pigments
5
200
(b)
150
Rainfall (mm)
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271
100
50
0
J
F
M
A
M
J
J
A
S
O
N
D
Figure 1. (a) Monthly mean air temperature and (b) monthly total
rainfall, through the study period. Error bars indicate the standard
error of the mean (n ¼ 28–31 days; values are averages of three
plots).
perchloric acid, and the mixture was centrifuged
(5000 rpm, for 20 min) at 48C. The pH was adjusted
to 7 by adding 300 – 400 ml of a sodium carbonate
solution. Reduced ASC/DHA content was determined using the spectrophotometer method
described by Takahama and Oniki (1992). Reduced
ASC content was determined by measuring the
absorbance at 265 nm (1 ¼ 14 mM21 cm21) after the
addition of ASC oxidase (1 U ml21) whereas DHA
content was determined by measuring the absorbance at 265 nm again following the addition of
2 mM DL-dithiothreitol (DTT).
For the measurements of enzyme activities, leaf
tissues were homogenized with 0.1 M phosphate
buffer pH 7.8 in a pre-chilled mortar. The homogenate was centrifuged at 48C for 20 min at 5000 rpm.
Ascorbate peroxidase (APX) activity was determined
spectrophotometrically by monitoring the decrease
in reduced ASC at A265 (1 ¼ 14 mM21 cm21) as
described by Nakano and Asada (1981). Activity was
expressed as mmol ASC min21 mg protein21. Glutathione reductase (GR) was assayed by monitoring the
glutathione-dependent oxidation of NADPH at
340 nm (Tietze 1969). The activity was expressed as
mmol GSH min21 mg21 protein. CAT activity was
Xanthophyll cycle pigments (VZA), Chl a, Chl b,
lutein, and b-carotene were determined according to
Munné-Bosch and Alegre (2000). Briefly, leaves
were repeatedly extracted (three times) with ice-cold
85% (v/v) acetone and 100% acetone using
sonication for 45 min at 48C (ultrasonic bath Typ
T570/H; Elma). Pigments were separated on a
Dupont nonendcapped Zorbax ODS-5 mm column
(250 £ 4.6 mm, 20% C; Scharlau, Barcelona, Spain)
at 308C at a flow rate of 1 ml min21. The solvents
consisted of (A) acetonitrile –methanol (85:15, v/v)
and (B) methanol–ethyl acetate (68:32, v/v). The
HPLC 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, Foster City,
CA, USA). Compounds were identified by their
characteristic spectra and by coelution with authentic
standards, which were purchased from Fluka.
Statistical analyses
Analyses of variance with gas exchange and
biochemical variables as dependent variables (one
mean value per plot; n ¼ 4) and treatment and
season as independent factors, and regression
analyses were conducted using STATISTICA v. 6.0
for Windows (StatSoft, Inc., Tulsa, OK, USA).
Statistical differences between treatments in each
season were also analyzed with a Student t-test.
Differences were considered significant at a probability level of p , 0.05.
Results
Environmental data
Soil humidity (v/v) in spring was 0.22 ^ 0.003 and
0.19 ^ 0.011 in control and drought plots, respectively. Whereas, in summer, the soil humidity was
0.13 ^ 0.011 and 0.12 ^ 0.003 in control and
drought plots, respectively. Thus, drought treatment
reduced the soil moisture by 10% on average in
relation to control plots, irrespective of the season.
Rainfall during the 3 months preceding measure-
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272
I. Nogués et al.
Figure 2. (a) Midday water potential (MPa), (b) midday maximal photochemical efficiency (Fv/Fm), (c) net photosynthetic rates
(mmol CO2 m22 s21), and (d) stomatal conductance (mmol H2O m22 s21) measured in the morning of Q. ilex leaves for control (C) and
drought (D) treatments during the spring and summer periods. Mean ^ SE (n ¼ 4 means of three measures for each plot) is shown.
Differences between means of control and drought-treated leaves are shown. *p , 0.05. Differences between means in spring and summer
for each of the treatments separately are also shown. þ þ þ p , 0.001; þ þ p , 0.01; þp , 0.05.
ments was 116 mm (February –April) and 78.4 mm
(May– July) (Figure 1). This last period, especially
May, was thus unusually wet. Irradiance
values during gas exchange measurements were
between 950 and 1050 mmol m22 s21 in April,
and between 1000 and 1450 mmol m22 s21 in July.
The leaf temperature during these measurements
ranged from 19 to 308C in April, and from 34 to 428C
in July.
Water potential (c)
The water potential (c) values were approximately
30% lower in drought-treated plants than in control
plants, in both seasons (Figure 2(a)). All values were
significantly more negative in summer than in spring
(Figure 2(a)).
Fluorescence
No significant differences between treatments were
found in Fv/Fm values in either spring or summer.
Irrespective of the treatment, Fv/Fm values were
significantly higher in summer than in spring (0.76
and 0.70, respectively) (Figure 2(b)).
Foliar photosynthetic rates (A) and stomatal
conductance (gs)
Net photosynthesis and stomatal conductance
decreased as a consequence of drought in spring
(44% and 27%, respectively) though only the
decrease in A was statistically significant. However,
no significant differences between treatments were
found in summer (Figure 2(c),(d)). A seasonal effect
was observed, as A and gs were significantly lower in
summer than in spring (Figure 2(c),(d)).
Isoprenoid emissions
Monoterpenes (a-pinene, b-pinene, and limonene)
were the main isoprenoids emitted by Q. ilex (Figure
3). Other monoterpenes (e.g. sabinene, b-myrcene,
and camphene) were also detected in the summer
sampling but the emissions were less than 10% than
the a-pinene, b-pinene, and limonene emissions.
Drought treatment did not alter significantly mono-
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Quercus ilex responses to field drought
273
Figure 3. (a) a-Pinene, (b) b-pinene, (c) limonene, and (d) total monoterpene emission rates (mg g21 h21) measured in the morning and
expressed per unit dry mass of Q. ilex leaves for control (C), and drought (D) treatments during the spring and summer periods. Mean ^ SE
(n ¼ 4 means of three measures for each plot) is shown. Differences between means of control and drought-treated leaves are shown.
***p , 0.001; **p , 0.01; *p , 0.05. Differences between means in spring and summer for each of the treatments separately are also shown.
þþþ
p , 0.001; þ þ p , 0.01; þp , 0.05.
terpene emission rates by Quercus leaves either in
spring or in summer. However, a clear seasonality
was found in the emission rates. Total monoterpene
emission of control Quercus leaves ranged between
practically zero in spring and 3.6 mg g21 d.m. h21 in
summer.
Concentration of antioxidants and secondary metabolites,
and activity of antioxidants
Metabolite concentrations are reported in the left
panels of Figure 4(a) – (c). Total phenolic compounds increased under drought by 11% in spring
and by 14% in summer (Figure 4(a)). Drought also
caused an increase of approximately 30% in total
ASC levels in both spring and summer samples
(Figure 4(b)). However, the redox state of ASC did
not change in drought-treated samples compared
with that in control samples (Figure 4(c)).
When comparing metabolite concentrations in
the two seasons, the concentration of phenolic
compounds was around 30% higher in summer
than in spring for both control and drought-treated
plants. Total ASC levels, however, decreased slightly
from spring to summer in both control and droughttreated plants (Figure 4(a),(b)).
The enzymatic activity of antioxidants is reported
in the right panels of Figure 4(d) –(f). CAT activity
was not altered by the drought treatment during
springtime, whereas it was enhanced 26% in summer
(Figure 4(d)). APX and GR activities, however,
resulted to be enhanced by drought in both spring
and summer. APX activity increased by 89% in
spring and 53% in summer, whereas GR activity
increased by 55% in spring and by 44% in summer
(Figure 4(e),(f)).
As for the seasonal effect, CAT, APX, and GR
activities were, respectively, 2.5-, 2.2-, and 1.9-fold
higher in summer than in spring (Figure 4(d) –(f)) in
control plants, and those activities were, respectively,
3-, 1.4-, and 1.4-fold higher in summer than in
spring for drought-treated plants.
Photosynthetic pigments
We did not find any significant difference in total Chl
(Figure 5(a)), Chl a/b ratio (Figure 5(b)), lutein and
b-carotene (Figure 5(c),(d)), or xanthophylls (Figure
5(e),(f)) between drought-treated and control plants.
However, important seasonal differences could be
observed. Total Chl in summer was approximately
70% higher than that in spring (Figure 5(a)). Also
274
I. Nogués et al.
(a)
(d)
100
Total ascorbate (µmol g[DW]–1)
(c)
CAT activity
(µmol min–1 mg–1 protein)
100
80
60
40
20
(e)
0
14
2
0
(f)
12
10
8
6
4
GR activity
(µmol min–1 mg–1 protein)
0.4
0.2
0.0
80
60
40
20
0
0.3
0.2
0.1
0.0
0.04
0.6
DHA/total ascorbate
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(b)
120
APX activity
(µmol min–1 mg–1 protein)
Phenolics
(mg gallic acid g [DW]–1)
140
C
D
Spring
C
D
Summer
0.03
0.02
0.01
0.00
C
D
Spring
C
D
Summer
Figure 4. (a) Phenolic compounds (mg of GAE g21), (b) total ascorbate (ASC) (mmol g21), (c) DHA/total ASC ratio, and activities of (d)
CAT, (e) APX, and (f) GR, all expressed in mmol min21 mg21 protein in Q. ilex control leaves (C) and in leaves exposed to drought (D)
during the spring and summer periods. Concentrations are expressed per unit dry mass. Mean ^ SE (n ¼ 4 means of three measures for
each plot) is shown. Differences between means of control and drought-treated leaves are shown. ***p , 0.001; **p , 0.01; *p , 0.05.
Differences between means in spring and summer for each of the treatments separately are also shown. þ þ þ p , 0.001; þ þ p , 0.01; þp , 0.05.
lutein and b-carotene were ca. three times higher in
summer than in spring (Figure 5(c),(d)). Chl a/chl b
ratio was 34% lower in summer than in spring
(Figure 5(b)). The de-epoxidation state of the
xanthophyll cycle (DPS) was higher in spring (ca.
0.80) than in summer (0.65), though this difference
was only slightly significant in control plants (Figure
5(f)).
Discussion
An inhibition of photosynthesis was expected in
plants suffering from water stress, mainly as a
consequence of the increased resistance to CO2 in
both the stomata and the mesophyll (Schulze & Hall
1982; Ogaya & Peñuelas 2003; Vitale et al. 2011).
However, in our experiment we could observe a
significant reduction in photosynthesis due to
drought only during the spring period. During
summer, photosynthesis of both control and
drought-treated plants decreased, and the droughtinduced inhibition of photosynthesis was not
statistically significant. Therefore, photosynthesis of
holm oak may be more affected in spring than in
summer, when subjected to predicted future
reduction in rainfall in the Mediterranean (IPCC
2007). The analysis of Chl fluorescence measurements yielded a different vision of drought impact on
primary metabolism with respect to gas exchange
measurements. In particular, the maximal efficiency
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Quercus ilex responses to field drought
275
Figure 5. (a) Chl a þ b (Chl) (mg g21), (b) Chl a/Chl b ratio, the lipophilic antioxidants (c) lutein (Lut) and (d) b-carotene (b-car) (mg g21),
(e) total xanthophylls cycle components (VZA) (mg g21), and (f) the DPS, given as (Z þ 0.5A)/VZA in Q. ilex control (C) leaves and in leaves
exposed to drought (D) during the spring and summer periods. Concentrations are expressed per unit dry mass. Mean ^ SE (n ¼ 4 means of
three measures for each plot) is shown. Differences between means in spring and summer for each of the treatments separately are shown.
þþþ
p , 0.001; þ þ p , 0.01; þp , 0.05.
of PSII (as indicated by the fluorescence parameter
Fv/Fm) was lower in spring than in summer,
indicating a possible occurrence of photoinhibition
(Powles 1984; Adams et al. 2004), perhaps induced
by cooler temperatures and bright days during
spring. Furthermore, the lower availability of photoprotective pigments, lutein, and b-carotene might
have not allowed efficient defense against lightinduced stress in spring, partially compensated by
the higher DPS. The fluorescence dataset also
suggests that the inhibition of photosynthesis in
summer was not caused by photochemical
limitations.
It has been reported that severe drought
decreases isoprenoid emission rates (Delfine et al.
2005; Peñuelas & Staudt 2010), but mild drought
often does not induce any change in emissions
(Pegoraro et al. 2004). The reduction in isoprenoid
emission seems to occur only when the severity of the
stress largely inhibits photosynthesis (Staudt et al.
2002; Brilli et al. 2007). A clear seasonality of
monoterpene emission was found, which was also
reported in previous studies (Llusià & Peñuelas
2000). This seasonality might be largely attributed to
higher summer temperature, as monoterpene emission by holm oak is largely temperature dependent
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276
I. Nogués et al.
(Loreto et al. 1996). Moreover, as photosynthetic
rates decreased from spring to summer, in agreement
with other authors (Llusià et al. 2011), the observed
increase in monoterpene emission rates in the same
period could not be explained in terms of substrate
availability. Thus, we conclude that temperature was
the main factor controlling monoterpene emission
rates over the studied seasons.
We could not observe any significant change in total
monoterpene emission due to drought. Interestingly,
however, when considering the three major individual
emitted monoterpenes, two opposite trends were
observed in the two seasons. In spring, there was a
tendency of a-pinene and b-pinene to increase in
drought-treated plants, compensated by the disappearance of limonene. In summer, a-pinene and bpinene, as well as the total amount of emitted
monoterpenes, were slightly reduced in the droughttreated plants, whereas limonene emission appeared to
be stimulated. Other authors have already reported
that monoterpene emission rates in oaks were relatively
insensitive to mild drought (e.g. Lavoir et al. 2009).
However, our results suggest that the impact of
drought can vary with the season, and reveal a peculiar
behavior of limonene in response to drought. The
effect of drought on limonene should be considered
when attempting to use monoterpenes as chemotaxonomic markers in oaks (Loreto et al. 2009) and
possibly also in other genera (Michelozzi et al. 2008).
The other component of the antioxidant system
that may actively protect the photosynthetic apparatus against photochemical damage is based on
metabolites and enzymes scavenging ROS. ROS
concentration must be tightly regulated by enzymatic
and nonenzymatic antioxidants (Asada 1999) to be
able to activate stress defense/acclimation responses.
We found a sustained increase in several nonenzymatic (total ASC, phenolic compounds) and
enzymatic (APX and GR activities) antioxidants,
both in spring and in summer, in drought-treated
plants. CAT activity, however, seemed to be mainly
under a strong seasonal influence. The response of
antioxidants to drought is species specific, and also
depends on the developmental and metabolic state of
the plant, and on duration and intensity of the stress
(Smirnoff 1993; Castillo 1996). For instance, APX
activity increased in tepary bean (Türkan et al. 2005)
and olive (Sofo et al. 2007) but decreased in sorghum
plants (Zhang & Kirkham 1996) under drought stress.
CATactivity was reported to increase, decrease, or not
change in response to drought (Smirnoff 1993; Zhang
& Kirkham 1996; Sofo et al. 2007). Under these
circumstances, it is difficult to detect a cause–effect
relationship between the induction of higher levels of
antioxidative defenses and the degree of drought
tolerance (Türkan et al. 2005). In our experiment, the
induction of phenolic compounds, total ASC content,
and antioxidant enzymes activities in plants experiencing drought, irrespective of the season, may indicate
the activation of defense responses aiming at scavenging ROS produced under increasing limitations of
primary metabolism, and to ultimately avoid stronger
oxidative damage.
The low concentration of phenolic compounds,
and the low activity of APX, GR, and CAT in spring
might also be of interest. We surmise now that a lower
availability of antioxidants might have in turn
contributed to make oak leaves sensitive to drought
in spring. Indeed, despite a drought-induced stimulation of enzymatic and non-enzymatic antioxidants
(with the exception of CAT), the levels of antioxidant
remained generally lower in spring than in summer in
drought-treated leaves. A seasonal variation in antioxidant protection has been reported in previous works
(Anderson et al. 1992; Vuleta et al. 2010). Although it
cannot be excluded that differences in the measured
parameters between the two studied seasons might be
due to the fact that leaves measured in spring were 1
year old, whereas those measured in summer were less
than 6 months old, this seems unlikely since in all cases
leaves were fully developed and mature.
In conclusion, though predicted future drought
conditions do not seem to severely alter the
physiology of Q. ilex, they may reduce photosynthesis
in spring, possibly due to a low pigment concentration and to an insufficient antioxidant protection,
associated with high diffusive limitation of CO2. In
summer, the increased resistance to CO2 entry
reduces photosynthesis in all oak leaves, but drought
is not expected to cause further reduction in
photosynthesis, as the photosynthetic apparatus of
oak leaves is fully protected by high pigment and
antioxidant levels. Moreover, the fact that plant
responses to stress depend on several factors
including plant species, age of plants, and previous
stress episodes as well as the interaction of drought
with other changeable environmental factors in the
field will make predictions about the net response of
plants to drought even more complicated.
Acknowledgments
This research was supported by funding from
Spanish Government grants CGL2006-04025/
BOS, CGL2010-17172/BOS and Consolider-Ingenio Montes (CSD2008-0040) and from the Catalan
Government grant SGR2009/458.
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