Tree Physiology 24, 813–822
© 2004 Heron Publishing—Victoria, Canada
Stomatal limitation to CO2 assimilation and down-regulation of
photosynthesis in Quercus ilex resprouts in response to slowly
imposed drought
KAREN PEÑA-ROJAS,1 XAVIER ARANDA2 and ISABEL FLECK1,3
1
Departament de Biologia Vegetal, Unitat Fisiologia Vegetal, Facultat Biologia, Universitat de Barcelona, Diagonal 645, E-08028, Barcelona, Spain
2
Present address: Departament de Tecnologia Hortícola, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Ctra. de Cabrils s/n, 08348,
Cabrils
3
Corresponding author ([email protected])
Received October 6, 2003; accepted December 21, 2003; published online May 3, 2004
Summary Holm oak (Quercus ilex L.) is native to hot, dry
Mediterranean forests where limited water availability often
reduces photosythesis in many species, and forest fires are frequent. Holm oaks resprout after a disturbance, with improved
photosynthetic activity and water relations compared with unburned plants. To better understand the role of water availability in this improvement, watering was withheld from container-grown plants, either intact (controls) or resprouts after
excision of the shoot, to gradually obtain a wide range of soil
water availabilities. At high water availability, gas exchange
rates did not differ between controls and resprouts. At moderate soil dryness, net photosynthesis of control plants decreased
as a result of increased stomatal limitation, whereas gas exchange rates of resprouts, which had higher midday and predawn leaf water potentials, were unchanged. Under severe
drought, resprouts showed a less marked decline in gas exchange than controls and maintained photosystem II integrity,
as indicated by chlorophyll fluorescence measurements. Photosynthesis was down-regulated in both plant types in response
to reduced CO2 availability caused by high stomatal limitation.
Lower non-stomatal limitations in resprouts than in control
plants, as evidenced by higher carboxylation velocity and the
capacity for ribulose -1,5-bisphosphate regeneration, conferred greater drought resistance under external constraints similar to summer conditions at midday.
Keywords: chlorophyll fluorescence, gas exchange, holm oak,
water relations.
Introduction
During summer, Mediterranean forests, which receive little or
no precipitation, are often subjected to severe drought as a
result of high temperature, irradiance and vapor pressure deficit
(VPD). Leaves exhibit decreases in relative water content (RWC)
and water potential (Ψ) (Quick et al. 1992, Savé et al. 1999).
Under these conditions, limited water availability reduces
photosynthesis in many species (Tenhunen et al. 1987, Chaves
1991). Stomatal closure, which diminishes carbon dioxide
(CO2) availability to mesophyll cell chloroplasts, has been described as the main limiting factor for CO2 assimilation in response to mild water stress (Lawlor and Cornic 2002). However, there is evidence that non-stomatal limitation inhibits
CO2 metabolism (Escalona et al. 1999, Flexas et al. 2002,
Lawlor and Cornic 2002, Maroco et al. 2002).
During a drought, absorption of light is likely to exceed that
required for photosynthetic assimilation and can lead to photoinhibition of the photosynthetic apparatus (Quick et al. 1992,
Méthy et al. 1996). Increased photorespiration serves as a sink
for excess excitation energy (Cornic and Briantais 1991, Osmond and Grace 1995). In addition, other processes, including
dissipation of light energy as heat (Demmig-Adams and Adams 1992, Horton et al. 1996, Gilmore 1997) and detoxification of activated oxygen by scavenger systems (Foyer and
Harbinson 1994) protect the photosynthetic apparatus in many
species, including holm oak (Fleck et al. 2000, El Omari et al.
2003a).
Climate models predict increased air temperature and potential evapotranspiration for the Mediterranean basin, together with decreased rainfall (Houghton 1997), which may
increase the risk of drought. These conditions increase not
only plant water stress, but the incidence of fire. Increased fire
frequency can lead to the exhaustion of reserves in some
species, thereby hindering the capacity for resprouting and
growth (Reich et al. 1990, Kruger and Reich 1997). Holm oak
(Quercus ilex L.) and cork oak (Quercus suber L.), the main
evergreen oak species in the Mediterranean basin, resprout after perturbation (Canadell et al. 1991, Retana et al. 1992).
Availability of water and nutrients is greater for resprouts than
for the original plants, because the root system of the resprouts
was originally associated with a much greater aerial biomass
(De Souza et al. 1986, Saruwatari and Davis 1989, Kruger and
Reich 1993). Shifts in shoot/root balance stimulate photosynthesis and rapid growth in Q. ilex (Fleck et al. 1996a, 1996b,
1998), Quercus rubra L. (Kruger and Reich 1993, 1997) and in
814
PEÑA-ROJAS, ARANDA AND FLECK
several chaparral species (Oechel and Hastings 1983, Hastings
et al. 1989, Reich et al. 1990).
The aim of our study was to describe and compare gas exchange responses during soil drying in leaves of undisturbed
holm oak seedlings and of seedlings resprouting after shoot
excision. Previous results obtained with resprouts of this species after fire (Fleck et al. 1998) or shoot excision (Fleck et al.
1996b) in the forest indicate increased photosynthetic activity
and improved water relations; however, these data were mostly obtained in the summer under severe drought conditions
(Fleck et al. 1996b, 1998). Moreover, resprouts and controls
did not share the same ground area, and so the results may have
been influenced by differences in the soil water content of
burned and unburned sites due to canopy transpiration. To
overcome these difficulties, we imposed a range of soil water
contents on potted plants grown in a covered nursery to ensure
the same irradiance, VPD and temperature for all plants. The
rate of water loss was adjusted so that it was similar for controls and resprouts. Previous experiments (Fleck et al. 1998,
2000, El Omari et al. 2003b) suggest that, under our experimental conditions, potted plants are subject to neither aerial
nor underground growth limitations.
To gain a detailed understanding of the resprouting process
and the survival capacity of holm oak after disturbances in
drought-prone areas, we sought to characterize the effects of
drought of varying severity on the water status and photosynthetic performance of holm oak plants. Specifically, we
measured net CO2 assimilation responses to intercellular CO2
or light to differentiate between stomatal and non-stomatal
contributions to decreases in photosynthesis that resulted from
drought in both plant types. Simultaneous measurements of
chlorophyll fluorescence provided data on changes in the efficiency of light utilization for electron transport and the occurrence of photoprotective mechanisms.
Materials and methods
Growth conditions and experimental design
In May, eighty 2-year-old holm oak plants (45.5 ± 1.7 cm
high, 71.0 ± 5.9 mm2 stem section, 164.5 ± 9.4 g DM m –2 leaf
mass per area (LMA) and 25.6 ± 3.2 g DM aerial biomass, on average) were lifted from a nursery and transferred to 9-l pots
filled with a mixture of local soil (Calcic Luvisol (FAOUnesco 1988) of texture: loam (0–60 cm depth), clay loam
(60 – 90 cm depth) and clay loam-silt loam (90 –150 cm
depth)) (40% by volume), peat (20%), vermiculite (20%) and
perlite (20%). Slow-release fertilizer (2 g l –1; 15:8:11 N,P,K +
2 Mg) was added to the substrate. The plants were placed in a
covered nursery under natural Mediterranean conditions in the
Experimental Fields of the University of Barcelona, NE Spain
(41°22′59″ Ν, 2° 6′44″ E, altitude 60 m above sea level) and
irrigated daily with 500 ml of water. In June, aerial biomass
was removed above the point of cotyledon attachment (cotyledonary node) of 40 randomly selected plants. The other 40
plants were left undisturbed as controls. In September, when
the excised plants began to resprout (six stems on average), all
pots were watered to container capacity (CC) and excess water
was allowed to drain freely. Thereafter, irrigation was withheld from half of the resprouts and half of the control plants to
induce gradual drought. The experimental design comprised
four groups: well-watered controls (C+), well-watered resprouts (R+), water-limited controls (C–) and water-limited
resprouts (R–). Pots from the irrigated treatments were watered to container capacity on alternate days. Pots containing
intact non-irrigated plants were supplemented with water to
match substrate water loss from pots containing non-irrigated
resprouts. To calculate water supplementation and to follow
soil water content (percentage container capacity (%CC)), five
pots per treatment were weighed at the beginning of the experiment and on alternate days thereafter.
Physiological measurements were made on five pots per
treatment about every 10 days for 2 months before watering
under conditions of light, temperature and VPD that simulated
those in the forest during summer (Fleck et al. 1998, 2000).
Withholding water for 60 days resulted in a wide range in the
degree of soil dryness.
Leaf gas exchange and chlorophyll fluorescence
Net photosynthesis (A) and stomatal conductance (gs) were
measured in the morning (M; 0930–1130 h), around midday
(Md; 1230–1500 h) and in the evening (Ev; 1730–1930 h) on
one attached and fully developed young leaf of eight plants per
treatment with a gas exchange system (LI-6400 Li-Cor, Lincoln, NE) equipped with a light source (6400-02B LED, LiCor). Environmental conditions in the chamber used for leaf
measurements were established in the M, at Md and in the Ev
as: photosynthetic photon fluence rate (PPFR) over the waveband 400–700 nm = 950, 1500 and 850 µmol m –2 s –1; ambient
water mole fraction = 19 ± 1, 17 ± 1 and 15 ± 1 mmol H2O
mol –1 air; leaf temperature = 28, 30 and 26 °C, respectively,
ambient CO2 concentration (Ca) = 350 µl l –1; and air flux =
150 µmol s – 1.
Every 10 days, light response curves (A/PPFR) and CO2 response curves of CO2 assimilation (A/Ci) were obtained for
each group of plants (one young leaf from four plants per treatment). For the former, Ca was set at 350 µl l –1, and for the latter,
PPFR was 600 µmol m –2 s –1, which was saturating for photosynthesis in holm oaks (see Figures 3a–c); Ci is the intercellular CO2 concentration. Data were recorded when measured parameters were stable (3 to 5 min). Inside the chamber,
the following conditions were maintained during the measurements: water mole fraction = 17 ± 1 mol H2O mol –1 air, leaf
temperature = 30 °C and air flow = 150 µmol s–1. Analyses of
the A/Ci curves allowed determination of changes in net CO2
assimilation at saturating Ci (Amax ), maximum carboxylation
velocity of Rubisco (Vcmax ), maximum potential rate of electron transport contributing to ribulose-1,5-bisphosphate
(RuBP) regeneration (Jmax ) and stomatal limitation (l) to light
saturating A for individual leaves. Parameters Vcmax and Jmax
were calculated by fitting a maximum likelihood regression
below and above the inflexion of the A/Ci response using the
Michaelis constants, Kc (CO2) and Ko (O2), as described by
McMurtrie and Wang (1993). Parameter l, which is the percent
TREE PHYSIOLOGY VOLUME 24, 2004
DROUGHT AND PHOTOSYNTHESIS IN HOLM OAK RESPROUTS
815
Figure 1. Vapor pressure deficit (VPD)
at midday (a) and percent container capacity (b and c) during drought treatment. In (c), values of well-watered
and water-limited resprouts have been
shifted to the previous day of measurement. Each value represents the mean
± SE of five measurements. Symbols:
䊉 = well-watered control (C+); 䊊 =
water-limited control (C–); 䊏 = wellwatered resprout (R+); and 䊐 = waterlimited resprout (R–). Values of R+
and C+ have been displaced slightly
for clarity. Arrows indicate the days of
measurement.
decrease in light-saturated net CO2 assimilation attributable to
stomata, was calculated as in Farquhar and Sharkey (1982).
Analyses of the A/ PPFR curves allowed determination of Asat,
the light-saturated rate of net CO2 assimilation at ambient CO2
concentration.
Steady-state modulated chlorophyll fluorescence was determined with a portable fluorimeter (Mini-PAM, Walz, Effeltrich, Germany), simultaneously with the M, Md and Ev gas
Figure 2. Morning, midday and evening net photosynthesis (A) (a, b
and c) and stomatal conductance (gs ) (d, e and f) versus percentage
container capacity. Each value represents the mean ± SE of eight measurements. For C+ and R+, data were the mean of all days, because
their %CC was maintained around 100% throughout the study. Symbols: 䊉 = well-watered control (C+); 䊊 = water-limited control (C–);
䉱 = well-watered resprout (R+); and 䉭 = water-limited resprout
(R–).
exchange measurements and during the plotting of A/PPFR
and A/Ci response curves. The Mini-PAM system was attached to a leaf chamber (LI-6400, Li-Cor) through a 2 mm
diameter plastic fiberoptic wire (MINI-PAM/F1) placed in the
Li-Cor chamber top adaptor for Mini-PAM (64400-10) in
combination with the Li-Cor light source. To this end, a support was specially designed to hold the light source 4 cm over
the chamber top adapter to allow the miniature fiber optics to
be positioned in such a way that no external light entered the
chamber. A rectangular, open-ended pipe with reflecting inner
faces was placed between the light source and the chamber top
to minimize light loss. Because the PPFR sensor of the light
source was used, a calibration curve was calculated to account
for light loss between the source and the chamber.
Light-adapted components of chlorophyll fluorescence
(steady-state yield (F), maximum fluorescence yield (Fm′ )
and quantum yield of photosystem II (PSII) photochemistry
(ΦPSII; equivalent to (Fm′ – F)/Fm′ )) (Genty et al. 1989) were
measured. Leaves were then dark-adapted to obtain minimum
fluorescence yield (Fo), maximum fluorescence yield (Fm) and
maximum quantum yield of PSII photochemistry (Fv /Fm)
(equivalent to (Fm – Fo)/Fm ). Adaptation took at least 20 min,
after which Fv /Fm values reach about 95% of the predawn values in Q. ilex (Fleck et al. 1998). Additional measurements
were taken at 0600 h to obtain predawn (PD) Fv /Fm, photochemical quenching of fluorescence (qP) (equivalent to (Fm′ –
F)/(Fm′ – Fo′ )) and the Fv′/Fm′ intrinsic efficiency of open PSII
centers during illumination (equivalent to (Fm′ – Fo′)/Fm′ ))
following Oxborough and Baker (1997). All data were corrected for changes in fluorescence detector sensitivity induced
by temperature variation in the Mini-Pam.
Leaf water status
We measured Ψ and leaf hydration (H ) at PD (0630 h), M
(0900 h), Md (1200 h) and Ev (1700 h) on one young leaf of
10 plants per treatment per day of measurement, and RWC
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
816
PEÑA-ROJAS, ARANDA AND FLECK
Table 1. Net CO2 assimilation at saturating intercellular CO2 concentration (Ci ) and light (Amax ), maximum carboxylation velocity of Rubisco
(Vcmax ), maximum potential rate of electron transport contributing to ribulose-1,5-bisphosphate (RuBP) regeneration (Jmax ), net photosynthesis at
350 µl l –1 Ci and saturating light (Asat ) used as a reference for the calculation of stomatal limitation (l) for each percentage container capacity
(%CC) and treatment (well-watered control (C+), well-watered resprouts (R+), water-limited control (C–) and water-limited resprouts (R–)).
Each value represents the mean ± SE of four measurements. For C+ and R+, data were the mean of all days, because their %CC was maintained
around 100% throughout the study. For C– under severe drought (35–30 %CC), it was impossible to calculate l because Asat at 350 µl l –1 Ci was
close to zero.
%CC
Treatment
C+
C–
R+
R–
Amax
(µmol m–2 s–1)
100–80%
60–40%
35–30%
10.2 ± 1.2
10.8 ± 2.5
9.1 ± 3.0
0.61 ± 0.2
10.5 ± 0.7
9.5 ± 0.4
9.7 ± 1.3
5.2 ± 0.4
Vcmax
(µmol m–2 s–1)
100–80%
60–40%
35–30%
29.1 ± 3.6
25.3 ± 7.2
20.3 ± 5.4
1.6 ± 0.3
32.9 ± 1.6
22.9 ± 0.3
22.8 ± 6.0
16.6 ± 0.3
Jmax
(µmol m–2 s–1)
100–80%
60–40%
35–30%
58.7 ± 6.8
58.0 ± 10.9
47.9 ± 12.8
4.3 ± 0.8
61.0 ± 2.9
48.9 ± 1.0
42.3 ± 10.7
26.6 ± 1.9
A sat at
350 µl l –1 Ci
(µmol m–2 s–1)
100–80%
60–40%
35–30%
7.8 ± 0.6
8.1 ± 0.4
5.4 ± 1.6
–
8.0 ± 0.3
6.1 ± 1.0
6.8 ± 1.3
3.6 ± 1.0
l (%)
100–80%
60–40%
35–30%
27.2 ± 6.7
28.6 ± 3.2
47.4 ± 8.4
–
31.1 ± 7.6
31.7 ± 7.0
38.8 ± 10.5
84.1 ± 6.7
only at PD. Leaf Ψ was measured with a Scholander-type pressure chamber (ARIMAD-2, Air-Far-Charuv Accessories, RamatHagoland, Israel). We calculated H as ((FM – DM)/DM)
and RWC was obtained as ((FM – DM)/(FSM – DM)100),
where FM is fresh mass, FSM is fresh saturated mass after rehydrating samples for 24 h in the dark) and DM is dry mass after oven-drying samples at 65 °C to constant weight.
Growth measurements
In June, after excision, the dry mass (DM) of the excised aerial
biomass (leaves and stems) was determined after drying to
constant weight in a forced air oven at 65 °C. At the end of the
study, plants from each treatment were harvested, separated
into plant components (leaves, stems and roots) and their DM
determined. Increases in leaf and stem biomass were calculated. At the beginning of the drought treatment and at the end
of study, basal diameter was measured with a caliper square
and stem section was calculated on 10 plants per treatment. To
assess leaf area and to calculate leaf mass per area (LMA), images were obtained with an Epson GT5000 scanner and processed with Scion Image for Windows 4.0.2 (Scion Corporation, Frederick, MD).
Statistical analysis
Plants were randomly selected for measurements (one young
leaf per plant) for each treatment and period. Analyses of variance (ANOVA) performed with SPSS for Windows v.11.0
(SPSS, Chicago, IL) were conducted to test for the main effects and interactions, against appropriate error terms, of per-
turbations (resprout versus control), water availability (irrigated versus non-irrigated), time of day (PD, M, Md, Ev) and
soil dryness (high %CC, intermediate %CC and low %CC) on
the parameters measured. For the analyses of Vcmax, Jmax and l,
data from each day were pooled. The A/Ci, A/PPFR and
ΦPSII/PPFR curves were analyzed by repeated ANOVA, where
Ci or PPFR was the intra-subject factor, and perturbations and
drought intensity were the inter-subject factors. Differences
were considered significant at P = 0.05. Owing to the high
number of factors (four) and potential interactions (15) in the
design, we did not differentiate significant differences indicated by ANOVA in the figures and tables; however, only significant differences are described in the text.
Results
Variations in mean VPD during the study are shown in Figure 1a. Figure 1b illustrates variations in substrate water loss
(%CC) in the various treatments. Well-watered conditions
were maintained for C+ and R+ throughout the experiment
and soil in the R– treatment reached a given %CC later than
soil in the C– treatment despite the water supplements. In general, soil in the R– treatment reached a given %CC one day of
measurement later than soil in the C– treatment (Figure 1c).
An initial period of constant substrate water loss was followed, both in controls and resprouts, by much lower rates of
water loss (Figure 1b). The turning point was 40 %CC for controls and 35 %CC for resprouts (30 and 54 days after the beginning of the experiment, respectively). Because the physiologi-
TREE PHYSIOLOGY VOLUME 24, 2004
DROUGHT AND PHOTOSYNTHESIS IN HOLM OAK RESPROUTS
Figure 3. Light response curves of CO2 assimilation (A/PPFR; a–c)
and light response curves of photosystem II (PSII) quantum yield
(ΦPSII /PPFR; d–f ) at three drought intensities (100–80, 60–40 and
35–30 %CC). Each value represents the mean ± SE of four measurements. For C+ and R+, data were the mean of all days, because their
%CC was maintained around 100% throughout the study. Symbols:
䊉 = well-watered control (C+); 䊊 = water-limited control (C–); 䉱 =
well-watered resprout (R+); and 䉭 = water-limited resprout (R–).
Abbreviations: PPFR = photosynthetic photon fluence rate over the
waveband 400–700 nm; %CC = percentage container capacity.
cal measurements differed significantly during the drought treatment, we distinguished three ranges of soil dryness: high %CC
(100–80%), intermediate %CC (60–40%) and low %CC (35–
30%).
Gas exchange
Plants in the C+ and R+ treatments showed constant and similar A and gs throughout the study with no daily variations (Figures 2a–f ). Our values for these parameters were similar to
those observed in forest studies (Fleck et al. 1996b, 1998) and
on potted plants after shoot excision (El Omari et al. 2003b).
Values of A and gs for R– and C– at 100–80 %CC were similar
to those for R+ and C+. As %CC declined to between 60 and
40 %CC (Figures 2a–f ), A and gs decreased in C– , whereas
they were maintained in R–. At the end of the study, when
817
Figure 4. Morning, midday and evening photosystem II (PSII) quantum yield (ΦPSII ) (a–c), maximum quantum yield of PSII (Fv /Fm )
(d–f ), photochemical quenching (qP) (g–i) and efficiency of energy
capture by open PSII reaction centers (Fv ′/Fm′) ( j– l) versus percentage container capacity (%CC). Each value represents the mean ± SE
of eight measurements. For C+ and R+, data were the mean of all
days, because their %CC was maintained around 100% throughout
the study. Symbols: 䊉 = well-watered control (C+); 䊊 = water-limited control (C–); 䉱 = well-watered resprout (R+); and 䉭 = waterlimited resprout (R–).
%CC was 35–30% (Figures 2a–f), A and gs showed a marked
decline in both C– and R–, but resprouts showed significantly
higher A and gs than controls at midday and especially in the
evening.
The A/Ci curves of C+ and R+ did not differ significantly at
the beginning of the study (Table 1). During the initial period
of soil drying (60–40 %CC), Amax was similar in C– and R–
and no differences were observed in well-watered plants. Only
when the %CC reached 35–30% did water-limited plants
show lower Amax than well-watered plants, especially C–. Val-
Table 2. Maximum quantum yield of photosystem II (Fv /Fm ) at predawn for each percentage container capacity (100–80, 60–40 and 35–30 %CC)
and treatment (well-watered control (C+), well-watered resprouts (R+), water-limited control (C–) and water-limited resprouts (R–)). Each value
represents the mean ± SE of eight measurements. For C+ and R+, data were the mean of all days, because their %CC was maintained around 100%
throughout the study.
%CC
100–80%
60–40%
35–30%
Treatment
C+
C–
R+
R–
0.80 ± 0.001
0.80 ± 0.001
0.81 ± 0.001
0.71 ± 0.003
0.80 ± 0.001
0.80 ± 0.001
0.82 ± 0.001
0.81 ± 0.001
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
818
PEÑA-ROJAS, ARANDA AND FLECK
as l increased; however we were unable to calculate l when
%CC = 35–30% because C– had very low A. In R–, l was similar between 100 and 40%CC and increased when %CC reached 35–30%. Photosynthetic responses to light (A/PPFR curves) showed that Asat was maintained at high soil water availability and at intermediate %CC (60–40%), with no differences between controls and resprouts (Figures 3a and 3b). At
35–30 %CC, the decline was markedly greater in C– than in
R– (Figure 3c).
Chlorophyll fluorescence
In well-watered plants, the values of chlorophyll fluorescence
parameters were maintained throughout the experiment and no
differences between C+ and R+ were detected. At low %CC
(35–30%), only C– showed a decline in ΦPSII (Figures 4a–c),
with values at midday and in the evening being 82 and 66%
lower, respectively, than in R–. Both Fv /Fm (Figures 4d–f) and
Fv /Fm at predawn (Table 2) were constant for R– throughout
the study, whereas at 35–30 %CC, C– showed significant decreases in Fv /Fm at midday, in the evening and at predawn (21,
31 and 12%, respectively). At low %CC, qP in C– declined at
midday and in the evening (a 65 and 40% decrease, respectively, compared with R–) (Figures 4g–i), whereas no significant changes in R– were observed during the study. Under
conditions of low %CC, Fv′/Fm′ declined in C– in the evening
(38% lower than R–) (Figures 4j–l ). In R–, no significant
changes were observed in this parameter throughout the study.
Responses of ΦPSII to changing PPFR (Figures 3d–f ) differed
between C– and R– only at low %CC. Under all PPFR conditions, C– had lower ΦPSII values than R–.
Figure 5. Predawn (PD), morning (M), midday (Md) and evening
(Ev) leaf water potential (Ψ) (a–d) and leaf hydration (H) (e–h) versus percentage container capacity (%CC). Each value represents the
mean ± SE of 10 measurements. For C+ and R+, data were the mean
of all days, because their %CC was maintained around 100% throughout the study. Symbols: 䊉 = well-watered control (C+); 䊊 = waterlimited control (C–); 䉱 = well-watered resprout (R+); and 䉭 =
water-limited resprout (R–).
ues of Vcmax, Jmax and l were similar in C+ and R+ (Table 1)
throughout the study. At 60–40 %CC, no significant differences in Vcmax and Jmax between R– and C– were observed. At
35–30 %CC, these parameters clearly decreased in C–, where-
Water status
Leaf Ψ was similar in C+ and R+ and showed daily changes,
with higher values at predawn and in the evening (Figures 5a–d). At intermediate %CC, the daily variations in Ψ in
C– and R– were similar to those observed in well-watered
plants. At low %CC, R– and C– showed a marked decline
throughout the day, with R– having significantly higher values
than C–. Leaf hydration was always significantly higher in
resprouts than in controls, regardless of soil dryness (Figures 5e–h). Predawn RWC was significantly higher in resprouts throughout the study, regardless of the %CC (Table 3).
Values of RWC were maintained until intermediate %CC, after which they declined by about 32% in C– and by 9% in R–.
Table 3. Relative water content (RWC) at predawn for each percentage container capacity (100–80, 60–40 and 35–30 %CC) and treatment
(well-watered control (C+), well-watered resprouts (R+), water-limited control (C–) and water-limited resprouts (R–)). Each value represents the
mean ± SE of eight measurements. For C+ and R+, data were the mean of all days, because their %CC was maintained around 100% throughout
the study.
%CC
100–80%
60–40%
35–30%
Treatment
C+
C–
R+
R–
84.3 ± 1.2
80.5 ± 1.4
82.7 ± 1.3
55.8 ± 3.2
86.4 ± 1.2
82.8 ± 1.8
89.2 ± 0.9
78.3 ± 1.6
TREE PHYSIOLOGY VOLUME 24, 2004
DROUGHT AND PHOTOSYNTHESIS IN HOLM OAK RESPROUTS
819
Table 4. Increments in stem section and aerial biomass during the 60-day study, subterranean biomass and aerial to subterranean biomass ratio at
the end of the study. Leaf mass per area (LMA), mean leaf area and total leaf area at the beginning and end of the drought treatment. Treatments
were well-watered control (C+), well-watered resprouts (R+), water-limited control (C–) and water-limited resprouts (R–). Values are means ± SE
of 10 replicates per treatment.
Treatment
C–
C+
R–
R+
55.7 ± 4.8
100.2 ± 9.2
20.6 ± 4.9
22.8 ± 3.7
20.5 ± 1.2
12.1 ± 1.4
25.5 ± 0.5
18.9 ± 2.0
4.1 ± 0.5
6.6 ± 0.7
5.1 ± 0.7
6.3 ± 0.9
Subterranean biomass
(gDM plant –1)
39.8 ± 3.6
45.7 ± 3.2
11.8 ± 1.3
15.8 ± 3.0
Shoot/root
(gDM plant –1)
1.42 ± 0.2
1.58 ± 0.2
1.07 ± 0.1
0.81 ± 0.1
Stem section increment
(mm2 plant –1)
Aerial biomass increment
(gDM plant –1)
Stem
Leaves
Mean leaf area
(cm2 leaf –1)
Initial
End
4.74 ± 0.4
4.65 ± 0.5
4.87 ± 0.2
5.85 ± 0.3
4.64 ± 0.2
5.47 ± 0.3
4.02 ± 0.3
5.01 ± 0.4
Total leaf area
(cm2 plant –1)
Initial
End
903.6 ± 27.3
1569.6 ± 20.4
857.1 ± 9.2
2211.2 ± 98.2
210.1 ± 7.9
490.3 ± 16.0
197.6 ± 15.7
483.2 ± 21.6
LMA
(gDM m – 2)
Initial
End
177.0 ± 5.2
177.5 ± 2.3
180.8 ± 1.9
158.1 ± 7.3
147.0 ± 3.5
135.8 ± 4.8
153.0 ± 3.1
128.2 ± 5.7
Growth parameters
At the end of the study, the increase in stem section and aerial
(leaf + stem) biomass differed between C– and C+, whereas
these parameters were similar in R– and R+ (Table 4). The aerial biomass increment was 71.5% higher in controls than in
resprouts, and this difference was mainly attributable to the
greater increase in stems (67%) than in leaves (23%). In contrast, the increase in biomass in resprouts was 29% higher in
leaves than in stems. Root biomass was 68% higher in controls
than in resprouts at the end of the study. Aerial to subterranean
biomass ratio ((leaves + stems)/roots) was only 27% lower in
resprouts than in controls (Table 4) in spite of excision. Soil
drying had no effects on root biomass or aerial to subterranean
biomass ratio of either controls or resprouts (Table 4).
At the beginning of the experiment, C– had higher LMA
than resprouts. Water-limited plants showed higher LMA than
well-watered plants (43% higher in C– than in C+ and 24%
higher in R– than in R+), and controls showed higher LMA
(21%) than resprouts at the end of study. Although LMA was
maintained in water-limited plants, it diminished by 14% in
well-watered plants. In C–, leaf size had decreased by the end
of the study, whereas in C+, R+ and R–, leaf areas increased
(see MLA, Table 4). During the study, total leaf area increased
by about 60% in C+, R+ and R–, but by only 42% in C– (Table 4). Consequently, the final total leaf area of resprouts was
about one fourth that of well-watered controls.
Discussion
Undisturbed holm oak plants (C–) showed a decline in stomatal conductance (Figures 2d–f) in response to soil drying
even before the onset of severe drought. Plants avoided desicca-
tion through stomatal closure and maintained constant and elevated RWC values at intermediate %CC, as reported elsewhere (Gulías et al. 2002, Medrano et al. 2002). Our results
reflect the conservative use of water in this species (Epron and
Dreyer 1990, Peñuelas et al. 1998, Tognetti et al. 1998, Fotelli
et al. 2000). At 60–40 %CC, stomatal closure limited photosynthesis in C–, as evidenced by the l values (Table 1). Stomatal conductance declined in response to soil drying before
any change in leaf Ψ was observed. This response may be associated with the participation of root-generated chemical signals (Davies and Zhang 1991).
The shoot/root ratio in R– was lower than in C– (Table 4),
resulting in higher soil water availability per transpiring area,
thereby allowing a water spending strategy in R–. Increased
water loss, evidenced by maintenance of gs until 40 %CC, did
not affect the water status of R– because values of predawn
RWC (Table 3), Ψ (Figures 5a– d) and H (Figures 5e– h) were
not only maintained but higher than in C–. Higher gs (30% on
average) in R– for more than 40 days compared with C– was
associated with increased A and thus growth. In resprouts, the
increase in aerial biomass did not differ between well-watered
and water-limited plants, whereas in controls, aerial biomass
had decreased by 26.5% by the end of the experiment (Table 4).
Low %CC (35–30 %CC) decreased daily leaf Ψ, RWC and
H in all plants and, to a lesser extent, in R–. However, RWC
declined by about 9% in R– compared with about 32% in C–,
which was also reflected in the smaller size of the leaves (Table 4). The lower total leaf area of resprouts (one third of C–,
one fourth of C+, Table 4), resulting in a lower transpirational
demand than that of C– even at the same %CC, may account
for this relative maintenance of water status. Nevertheless, at
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
820
PEÑA-ROJAS, ARANDA AND FLECK
this drought intensity, R– showed stomatal closure, as indicated by lower gs, increased l and a marked decline in photosynthesis, especially at high irradiance and VPD (midday and
evening measurements), although all values were significantly
higher than in C–. Again, stomata seemed to respond before
RWC or leaf Ψ (Gulías et al. 2002, Medrano et al. 2002) to
changes in soil water status. Non-stomatal and stomatal limitations of photosynthesis occurred in both plant types. Decreases in
Asat (Figures 3a– c) and Amax (Table 1), which were accompanied
by reductions in Vcmax and Jmax (Table 1) in C– and R–, indicate a
down-regulation of CO2 assimilation to adjust mesophyll capacity to the decreased CO2 supply (Chaves et al. 2002, Lawlor and
Cornic 2002) caused by stomatal closure. Decreases in Vcmax
may result from reductions in the amount of active Rubisco.
The fall in Jmax is directly related to limited RuBP regeneration, which may be due to inadequate ATP or NADPH supply
or both, or low enzymatic activity of the PCR cycle (Lawlor
2002, Parry et al. 2002). The decline in Asat in R– was not correlated with changes in RWC, as reported for olive trees
(Nogués and Baker 2000). We found that R– had higher Vcmax
(90%) than C–, in agreement with a forest study in which holm
oak resprouts showed 45% higher Rubisco activity in summer
midday conditions than undisturbed vegetation (Fleck et al.
1996a). Under drought conditions, Ci may be overestimated
because of patchy stomatal closure (Laisk 1983) and the increased contribution of cuticular transpiration (Boyer et al.
1997). However, here we imposed drought slowly, which minimized or eliminated patchiness (Cornic and Massaci 1996).
Furthermore, the differences in A/Ci responses between treatments were too large to be explained by these effects.
Low %CC did not affect chlorophyll fluorescence parameters in resprouts (Figure 4), whereas controls showed decreases in ΦPSII at midday and in the evening (Figures 4a and 4c),
indicating down-regulation of electron transport (Chaumont et
al. 1995). In control plants, decreases in ΦPSII were accompanied by reductions in qP (number of open PSII centres) and
Fv′/Fm′ (Figures 4g–l). Decreases in Fv′/Fm′ are indicative of
increased excitation energy of quenching processes in the PSII
antennae (Horton et al. 1996), which protect the photosynthetic apparatus from photodamage. The participation of the
xanthophyll cycle (Demmig-Adams and Adams 1996) and a
lutein epoxide cycle, as reported for holm oak in the forest
(Llorens et al. 2002, García-Plazaola et al. 2002, 2003), may
have contributed to photoprotection (Fleck et al. 1998, 2000).
Lower Fv′/Fm′ and qP in controls were reflected in lower midday Fv /Fm values when Ψ reached –3.0 MPa. These results are
consistent with those reported in the field for Q. ilex (Méthy et
al. 1996). Resprouts maintained Fv /Fm values of 0.8 throughout the day (Figure 4 d–f ), indicating the lack of photodamage
to PSII centers. Chlorophyll fluorescence parameters in C–
and R– were less affected by drought than CO2 assimilation, as
reported for other Mediterranean plants (Epron et al. 1992,
Pereira and Chaves 1995, Méthy et al. 1996, Nogués et al.
2001) and are indicative of PSII stability under drought conditions.
In conclusion, photosynthesis was not enhanced in resprouts grown under well-watered conditions compared with un-
disturbed vegetation. With increasing soil dryness, the lower
water demand of resprouts, as a result of their lower total leaf
area, accounted for the maintenance of RWC and high gs and
A, whereas in undisturbed plants, at the same %CC, stomatal
closure limited photosynthesis. At low %CC (35–30%), nonstomatal limitations to photosynthesis were also less marked
in resprouts. These differences in photosynthetic responses
during drought are associated with higher carbon gain in resprouts than in undisturbed plants and may be responsible for the
enhanced performance after disturbances in drought-prone areas.
Acknowledgments
The research was supported by funds from the Generalitat de Catalunya 1999 SGR0020. We thank Dr. J. Flexas and Dr. S. Nogués for
helpful discussion, Dr. J. Casadesús, J. Matas and R. Simmonneau
(Servei de Camps Experimentals, Universitat de Barcelona) for technical assistance, and R. Rycroft (Servei d’Assessorament Lingüístic,
Escola d’Idiomes Moderns, Universitat de Barcelona) for correcting
the English manuscript. K. Peña-Rojas was the recipient of a doctorate grant from the AECI (Agencia Española de Cooperación con
Iberoamérica) and from the Faculty of Forestry Engineering, University of Chile.
References
Boyer, J.S., S.C. Wong and G.D. Farquhar. 1997. CO2 and water vapor exchange across leaf cuticle (epidermis) at various water potentials. Plant Physiol. 114:185–191.
Canadell, J., F. Lloret and L. López-Soria. 1991. Resprouting vigour
of two Mediterranean shrub species after experimental fire treatments. Vegetatio 95:119–126.
Chaumont, M., J.F. Morot-Gaudry and C. Foyer. 1995. Effects of
photoinhibitory treatment on CO2 assimilation, the quantum yield
of CO2 assimilation, D1 protein, ascorbate, glutathione and xanthophyll contents and the electron transport in vine leaves. Plant
Cell Environ. 18:1358–1366.
Chaves, M.M. 1991. Effects of water deficits on carbon assimilation.
J. Exp. Bot. 42:1–16.
Chaves, M.M., J.S. Pereira, J. Maroco, M.L. Rodrigues, P.P. Ricardo,
M.L. Osório, I. Carvalho, T. Faria and C. Pinheiro. 2002. How
plants cope with water stress in the field? Photosynthesis and
growth. Ann. Bot. 89:907–916.
Cornic, G. and J.-M. Briantais. 1991. Partitioning of photosynthetic
electron flow between CO2 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.) at different CO2 concentrations and during
drought stress. Planta 183:178–184.
Cornic, G. and A. Massaci. 1996. Leaf photosynthesis under drought
stress. In Photosynthesis and the Environment. Ed. N.R. Baker.
Kluwer Academic Press, Dordrecht, pp 347–366.
Davies, W.J. and J. Zhang. 1991. Root signals and the regulation of
growth and development of plants in drying soil. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 42:55–77.
Demmig-Adams, B. and W.W. Adams, III. 1992. Photoprotection and
other responses of plants to high light stress. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 43:599–626.
Demmig-Adams, B. and W.W. Adams, III. 1996. Xanthophyll cycle
and light stress in nature: uniform response to excess direct sunlight
among higher plant species. Planta 198:460–470.
De Souza, J., P.A. Silka and S.D. Davis. 1986. Comparative physiology of burned and unburned Rhus laurina after chaparral wildfire.
Oecologia 71:63–68.
TREE PHYSIOLOGY VOLUME 24, 2004
DROUGHT AND PHOTOSYNTHESIS IN HOLM OAK RESPROUTS
El Omari, B., I. Fleck, X. Aranda, A. Abadia, A. Cano and M.B.
Arnao. 2003a. Total antioxidant activity in Quercus ilex L. resprouts after fire. Plant Physiol. Biochem. 41:41–47.
El Omari, B., X. Aranda, D. Verdaguer, G. Pascual and I. Fleck.
2003b. Resource remobilization in Quercus ilex L. resprouts. Plant
Soil 252:349–357.
Epron, D. and E. Dreyer. 1990. Stomatal and non-stomatal limitation
of photosynthesis by leaf water deficits in three oaks species: a
comparison of gas exchange and chlorophyll a fluorescence data.
Ann. Sci. For. 47:435–450.
Epron, D., E. Dreyer and N. Bréda. 1992. Photosynthesis of oak trees
(Quercus petraea (Matt) Liebl.) during drought under field conditions: diurnal course of net CO2 assimilation and photochemical efficiency of photosystem II. Plant Cell Environ. 15:809–820.
Escalona, J.M., J. Flexas and H. Medrano. 1999. Stomatal and nonstomatal limitations of photosynthesis under water stress in fieldgrown grapevines. Aust. J. Plant Physiol. 26:421–433.
FAO-UNESCO. 1988. Soil maps of the world. Revised legend. Rome.
Farquhar, G.D. and T.D. Sharkey. 1982. Stomatal conductance and
photosynthesis. Annu. Rev. Plant Physiol. 33:317–345.
Fleck, I., D. Grau, M. Sanjosé and D. Vidal. 1996a. Carbon isotope
discrimination in Quercus ilex resprouts after fire and tree-fell.
Oecologia 105:286–292.
Fleck, I., D. Grau, M. Sanjosé and D. Vidal. 1996b. Influence of fire
and tree-fell on physiological parameters in Quercus ilex resprouts.
Ann. Sci. For. 53:337–346.
Fleck, I., K.P. Hogan, L. Llorens, A. Abadía and X. Aranda. 1998.
Photosynthesis and photoprotection in Quercus ilex resprouts after
fire. Tree Physiol. 18:607–614.
Fleck, I., X. Aranda, B. El Omari, J. Permanyer, A. Abadía and K.P.
Hogan. 2000. Light energy dissipation in Quercus ilex resprouts after fire. Aust. J. Plant Physiol. 27:129–137.
Flexas, J., J. Bota, J.M. Escalona, B. Sampol and H. Medrano. 2002.
Effects of drought on photosynthesis in grapevines under field conditions: an evaluation of stomatal and mesophyll limitations. Funct.
Plant Biol. 29:461–471.
Fotelli, M.N., K.M. Radoglou and H.I.A. Constatinidou. 2000. Water
stress responses of seedlings of four Mediterranean oak species.
Tree Physiol. 20:1065–1075.
Foyer, C.H. and J. Harbinson. 1994. Oxygen metabolism and the regeneration of photosynthetic electron transport. In Causes of Photooxidative Stress and Amelioration of Defence Systems in Plants.
Eds. C.H. Foyer and P.M. Mullineaux. CRC Press, Boca Raton,
FL, pp 1–42.
García-Plazaola, J.I., A. Hernández, E. Errasti and J.M. Becerril.
2002. Occurrence and operation of the lutein epoxide cycle in
Quercus species. Funct. Plant Biol. 29:1075–1080.
García-Plazaola, J.I., A. Hernández, J.M. Olano and J.M. Becerril.
2003. The operation of the lutein epoxide cycle correlates with energy dissipation. Funct. Plant Biol. 30:319–324.
Genty, B., J.M. Briantais and N.R. Baker. 1989. The relationship between the quantum yield of photosynthetic electron transport and
quenching of chlorophyll fluorescence. Biochim. Biophys. Acta
990:87–92.
Gilmore, A.M. 1997. Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves.
Physiol. Plant. 99:197–209.
Gulías, J., J. Flexas, A. Abadia and H. Medrano. 2002. Photosynthetic
responses to water deficit in six mediterranean sclerophyll species:
possible factors explaining the declining distribution of Rhamnus
ludovici-salvatoris, an endemic Balearic species. Tree Physiol. 22:
687–697.
821
Hastings, S.J., W.C. Oechel and N. Sionit. 1989. Water relations and
photosynthesis of chaparral resprouts and seedlings following fire
and hand clearing. In The California Chaparral/Paradigms Reexamined. Ed. S.C. Keeley. Natural History Museum of Los Angeles,
Los Angeles, CA, pp 107–113.
Horton, P., A.V. Ruban and R.G. Walters. 1996. Regulation of light
harvesting in green plants. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 4:655–684.
Houghton, J.T. 1997. Global warming: the complete briefing. 2nd
Edn. Cambridge University Press, Cambridge, 251 p.
Kruger, E.L. and P.B. Reich. 1993. Coppicing alters ecophysiology of
Quercus rubra sampling in Wisconsin forest openings. Physiol.
Plant. 89:741–750.
Kruger, E.L. and P.B. Reich. 1997. Response of hardwood regeneration to fire in mesic forest openings. II. Leaf gas exchange, nitrogen
concentration and water status. Can. J. For. Res. 27:1832–1840.
Laisk, A. 1983. Calculation of leaf photosynthetic parameters considering the statistical distribution of stomatal apertures. J. Exp. Bot.
34:1627–1635.
Lawlor, D.W. 2002. Limitation to photosynthesis in water-stressed
leaves: stomata vs. metabolism and the role of ATP. Ann. Bot. 89:
871–885.
Lawlor, D.W. and G. Cornic. 2002. Photosynthetic carbon assimilation and associated metabolism in relation to water deficits in
higher plants. Plant Cell Environ. 25:275–294.
Llorens, L., X. Aranda, A. Abadia and I. Fleck. 2002. Variations in
Quercus ilex chloroplast pigment content during summer stress: involvement in photoprotection according to principal component
analysis. Funct. Plant Biol. 29:81–88.
Maroco, J.P., M.L. Rodrigues, C. Lopes and M.M. Chaves. 2002.
Limitation to leaf photosynthesis in field-grown grapevine under
drought-metabolic and modelling approaches. Funct. Plant Biol.
29:451–459.
McMurtrie, R.E. and Y.-P. Wang. 1993. Mathematical models of the
photosynthetic responses of tree stands to rising CO2 concentrations and temperatures. Plant Cell Environ. 16:1–13.
Medrano, H., J.M. Escalona, J. Bota, J. Gulías and J. Flexas. 2002.
Regulation of photosynthesis of C3 plants in response to progressive drought: stomatal conductance as a reference parameter. Ann.
Bot. 89:895–905.
Méthy, M., C. Damesin and S. Rambal. 1996. Drought and photosystem II activity in two Mediterranean oaks. Ann. Sci. For. 53:
255–262.
Nogués, S. and N.R. Baker. 2000. Effects of drought on photosynthesis in Mediterranean plants grown under enhanced UV-B radiation.
J. Exp. Bot. 51:1309–1317.
Nogués, S., S. Munné-Bosch, J. Casadesús, M. López-Carbonell and
L. Alegre. 2001. Daily time course of whole-shoot gas exchange
rates in two drought-exposed Mediterranean shrubs. Tree Physiol.
21:51–58.
Oechel, W.C. and S.J. Hastings. 1983. The effects of fire on photosynthesis in chaparral resprout. In Mediterranean-Type Ecosystems.
Eds. F.J. Kruger, D.T. Mitchell and J.U.M. Jarvis. Ecol. Stud. 43.
Springer-Verlag, Berlin, pp 274–285.
Osmond, C.B. and S.C. Grace. 1995. Perspectives on photoinhibition
and photorespiration in the field: quintessential inefficiencies of the
light and dark reactions of photosynthesis. J. Exp. Bot. 46:
1415–1422.
Oxborough, K. and N.R. Baker. 1997. An instrument capable of imaging chlorophyll a fluorescence from intact leaves at very low
irradiance and at the cellular and sub-cellular levels of organization. Plant Cell Environ. 20:1473–1483.
Parry, M.A., P.J. Andralojc, S. Khan, P.J. Lea and A.J. Keys. 2002.
Rubisco activity: effects of drought stress. Ann. Bot. 89:833–839.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
822
PEÑA-ROJAS, ARANDA AND FLECK
Peñuelas, J., I. Filella, J. Llusià, D. Siscart and J. Piñol. 1998. Comparative field study of spring and summer leaf gas exchange and
photobiology of the Mediterranean trees Quercus ilex and Phyllyrea latifolia. J. Exp. Bot. 49:229–238.
Pereira, J.S. and M.M. Chaves. 1995. Plant responses to drought under climate change in Mediterranean-type ecosystems. In Global
Change and Mediterranean-Type Ecosystems. Eds. J.M. Moreno
and W.C. Oechel. Ecol. Stud. 117:140–160.
Quick, W.P., M.M. Chaves, R. Wendler et al. 1992. The effect of water
stress on photosynthetic carbon metabolism in four species grown
under field conditions. Plant Cell Environ. 15:25–36.
Reich, P.B., M.D. Abrams, D.S. Ellsworth, E.L. Kruger and T.J.
Tabone. 1990. Fire affects ecophysiology and community dynamics of central Wisconsin oak forest regeneration. Ecology 71:
2179–2190.
Retana, J., J. Riba, C. Castell and J.M. Espelta. 1992. Regeneration by
sprouting of holm oak (Quercus ilex) stands exploited by selection
thinning. Vegetatio 99/100:355–364.
Saruwatari, M.W. and S.D. Davis. 1989. Tissue water relations of
three chaparral shrub species after wildfire. Oecologia 80:
303–308.
Savé, R., C. Castell and J. Terradas. 1999. Gas exchange and water relations. In Ecology of Mediterranean Evergreen Oak Forests. Eds.
F. Rodà, J. Retana, C.A. Gracia and J. Bellot. Ecol. Stud. 137.
Springer-Verlag, Berlin, pp 135–147.
Tenhunen, J.D., R.W. Pearcy and O.L. Lange. 1987. Diurnal variations in leaf conductance and gas exchange in natural environments. In Stomatal Function. Eds. E. Zeiger, G.D. Farquhar and
I.R. Cowan. Stanford University Press, Stanford, pp 323–351.
Tognetti, R., A. Longobucco, F. Miglietta and A. Raschi. 1998. Transpiration and stomatal behaviour of Quercus ilex plants during the
summer in a Mediterranean carbon dioxide spring. Plant Cell Environ. 21:613–622.
TREE PHYSIOLOGY VOLUME 24, 2004