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Forest Ecology and Management 210 (2005) 117–129
Effects of the interaction between drought and shade on water
relations, gas exchange and morphological traits in cork oak
(Quercus suber L.) seedlings
I. Aranda a,*, L. Castro b, M. Pardos a, L. Gil b, J.A. Pardos b
a Instituto Nacional de Investigaciones Agrarias y Tecnologıas Agroalimentarias, Carretera Coruna Km., 7,5 28040 Madrid, Spainb Unidad de Anatomıa, Fisiologıa y Genetica Forestal, Escuela Tecnica Superior de Ingenieros de Montes,
Universidad Politecnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
Received 4 March 2004; received in revised form 22 November 2004; accepted 7 February 2005
Abstract
The combined effect of drought and light on different physiological and biochemical traits was assessed in cork oak (Quercus
suber L.) seedlings grown under two levels of light availability and submitted to a long-standing drought. Watering was
withdrawn after germination and seedlings were allowed to dry to a water content of ca. 50% of field capacity. At this point,
water-stressed seedlings were grown under moderate drought and two light regimes: high light (HL—50%) and low light (LL—
2%). Soil water in control plants was kept close to field capacity (90–100%) for both light environments. Water-relations
parameters derived from P–V curves, gas exchange and water status at predawn (Cpd) were evaluated at twice during the
experiment. Nitrogen and chlorophyll contents were determined in the same leaves used for the gas exchange measurements. In
addition, maximum rate of carboxylation (Vcmax) and electronic transport (Jmax) were derived from A–Ci curves in well-watered
seedlings.
The variation on moisture availability during the experiment was the same under both light environments. In control plants,
Cpd was over �0.3 MPa at the two harvests, while stressed seedlings decreased to �0.9 MPa, with no differences between light
treatments. Water stress decreased osmotic potentials at full (Cp100) and zero turgor (Cp0). The regressions between both
potentials and Cpd showed a higher intercept in shade grown seedlings. This fact will point out the higher osmoregulation
capacity in sun seedlings whatever water availability.
Nitrogen investment on a per leaf mass (Nmass), chlorophyll content (Chlmass) and SLA tended to show a typical pattern of
sun-shade acclimation. Thus, the three parameters increased with shade. Only for Nmass there was a significant effect of watering,
since water stress increased Nmass.
LL plants showed a lower photosynthetic capacity in terms of maximum net photosynthesis at saturating light (Amax), which
was related to a decrease in Vcmax and Jmax. Both parameters varied with specific leaf area (SLA) in a similar way. The low-light
environment brought about a higher nitrogen investment in chlorophyll, while under high-light environment the investment was
higher in carboxylation (Vcmax) and electronic transport ( Fmax).
* Corresponding author. Tel.: +34 1 3367113; fax: +34 1 5439557.
E-mail address: [email protected] (I. Aranda).
0378-1127/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.foreco.2005.02.012
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129118
Stomatal conductance to water vapour (gwv) and Amax were lower in low-light seedlings independently of watering. In
addition, there was a trend to keep higher intrinsic water use efficiency (IWUE) under high light environment. The increase of
IWUE under water stress was higher in HL seedlings. This was as consequence of the steeper decline in gwv as Cpd decreased.
The decrease of Amax with Cpd occurred in a similar way in LL and HL seedlings. Thus, the HL seedlings tended to sustain a
higher ability to increase IWUE than LL seedlings when they were submitted to the same water stress.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Cork oak; Drought; Light; Photosynthesis; Osmotic adjustment; Water use efficiency
1. Introduction
The interactive effects of shade and drought in
growth and physiology of seedlings have been
previously described (Gauhl, 1979; Chapin et al.,
1987; Holmgren, 2000; Valladares and Pearcy, 2002).
The impact of such interaction is of major interest for
understanding regeneration success of forest tree
species in Mediterranean ecosystems. Competition for
water in stands dominated by mature trees or shrubs
may exacerbate drought effects on tree seedlings
established in the understory (Burton and Bazzaz,
1995; Valladares and Pearcy, 2002). The ability of
regenerated seedlings to respond to drought under
shade conditions is also recognized to influence
patterns of forest distribution even in relatively
aseasonal climatic zones, such as tropical rain forest
(Fisher et al., 1991; Newbery et al., 1999; Gibbons and
Newbery, 2002). Furthermore, the facilitation of shade
by pre-existing vegetation, which favours forest tree
species recruitment, seems a general rule, despite the
possible interspecific competition for water and other
resources (see Callaway, 1995; Callaway et al., 2003).
Although the above-mentioned issue has been
profusely studied in terms of changes in biomass
allocation and relative growth rate (Burslem et al., 1996;
Sack and Grubb, 2002; Sack et al., 2003), there is little
information about the physiological response to the
interaction of drought and shade (Abrams and
Mostoller, 1995; Valladares and Pearcy, 1997, 2002;
Aranda et al., 2001). In some cases, patterns shown in
the literature are related to the particular conditions of
the study. Confuse and sometimes divergent results do
not allow us to establish the general role of drought in the
performance of seedlings grown under deep shade
(Veenendaal et al., 1996; Poorter and Hayashida-Oliver,
2000). Sack and Grubb (2002) have recently summar-
ized five possible hypotheses about the trade-off
between shade tolerance and drought tolerance. One
of the hypothesis is based on the model developed by
Smith and Huston (1989). Furthermore, Holmgren et al.
(1997) established a model of facilitation, in which light
and water availabilities were taken as main variables in
the process. One of the outputs from the model was that
facilitation under shade conditions takes place only
when water availability overcomes the limitation
imposed by shade in seedling’s carbon balance.
The interaction of light and water stress may be a
compromise between contradictory patterns of seed-
ling’s physiological response. For instance, lower
osmotic adjustment ability in leaves grown under
increasing shade conditions within the canopy has
been reported (Uemura et al., 2000; Niinemets, 2001);
as well as in seedlings established under low-light
environments (Ellsworth and Reich, 1992; Abrams
and Mostoller, 1995; Gebre et al., 1998; Tschaplinski
et al., 1998; Delperee et al., 2003). On the other hand,
it is widely recognized that osmotic potential at full
turgor (Cp100) may decrease because of an active
metabolic accumulation of osmolytes when seedlings
of drought-tolerant species are exposed to water stress
(Collet and Guehl, 1997). Thus, the occurrence of dry
periods under shade conditions might comprise an
awkward situation between shade- and drought-
tolerance. This compromise is shown by the lower
ability to develop mechanisms of drought-tolerance,
such as osmotic adjustment, under low irradiances
(Auge et al., 1990; Abrams and Mostoller, 1995;
Aranda et al., 2001). In this sense, it is important to
emphasize the ability of seedlings to adjust their
physiological response under low light and water-
limitant conditions, which in many cases are acting in
concert (Mulkey and Pearcy, 1992). These concurrent
environmental conditions are usually found in the
understory of Mediterranean forests during the
summer months. Probably, they are among the main
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129 119
factors limiting forest regeneration and long-term
recruitment of seedlings in the understory.
Osmotic adjustment as a mechanism of water-stress
tolerance is generally limited to sunny sites, where the
negative effects of drought are usually more intense
(Kloeppel et al., 1993), although not always (Valla-
dares and Pearcy, 2002). In these cases, the higher
photosynthetic capacity of seedlings grown under high
irradiance allows an active accumulation of osmo-
lytes, such as soluble sugars (Ellsworth and Reich,
1992). Thus, the ability to set in mechanisms such as
osmotic adjustment may be conditioned by the
development of a higher photosynthetic capacity
under sunny environments (Abrams, 1988; Kloeppel
et al., 1993; Mendes et al., 2001).
The difficulty on interpreting the results about the
interaction between light and drought arises when
plants growing under various irradiances are sub-
mitted to different degrees of water stress (Kolb et al.,
1990; Robakowski et al., 2003). Trying to overcome
these shortcomings, a greenhouse study was carried
out to determine the changes in water relations and gas
exchange of cork oak seedlings submitted to the same
soil water availability and growing under shade or
sunny conditions. Two questions were arised: first, if
deep shade decreased seedlings’ ability to develop
physiological mechanisms of water stress tolerance.
Second, if water lost is regulated in a similar manner
on seedlings submitted to the same water stress, but
grown under different light environments. The main
objective was to analyse how the ability of cork-oak
seedlings to respond to a moderate drought may be
altered under low light regimes.
2. Materials and methods
Ripe acorns were collected from 20 trees in a cork
oak woodland (dehesa) at Alburquerque (South-
western Spain) and kept at 2–4 8C for 3 months.
Two hundred acorns were placed in a seedbed inside a
climatic chamber at 25 8C for germination. After 2
weeks, when the radicule was 2 cm long, sixty acorns
were selected and transplanted to plastic rhizotrons
25 cm � 5 cm surface and 100 cm length. Rhizotrons
were placed in an iron framework, so that the
structures were tilted at 308 from the vertical. Growing
medium consisted of a peat-sand (3:1, v/v) mixture to
which a 6 months slow release fertilizer (N:P:K,
20:10:20 + micronutrients) was added (3 g l�1). Seed-
lings were grown in a heated greenhouse within a daily
range of 15–30 8C.
Two light environments, replicated in two blocks,
were tested (LL—low light, 2% and HL—high light,
50% of full sunlight). A shade-cloth covered the
seedlings in the LL treatment. Seedlings were subjected
to one of two watering regimes: well-watered (W) and
stressed seedlings (S). Soil moisture was recorded
weekly by time domain reflectometry (TDR). Three
small windows, 25 cm apart, were opened on the back of
the rhizotrons for moisture measurements along the
growing medium. One additional measurement was
taken in the surface of the substrate. This protocol
allowed to integrate soil moisture content from the
whole rooting zone and to sustain a water soil moisture
gradient from the top to the bottom of the soil in a similar
way to natural environments. Rhizotrons within a block
were randomly rotated every week.
Water-stressed seedlings (S) were allowed to dry to
a soil moisture of ca. 15% in the rizosphere (reached
100 days after the beginning of the water treatment).
To assure the same rate of imposition of the water
stress conditions independently of the light environ-
ment, seedlings in the HL treatment were irrigated
with the same volume of water lost by LL-seedlings
between two irrigations. When the 15% soil moisture
was reached, a gradient of water availability was
maintained in minirhizotrons which brought about the
substrate to 50–60% field capacity. Afterwards, soil
moisture content average (15%) was maintained
during 93 days in LLS and HLS seedlings by adding
water through the windows made in the rhizotrons. All
well-watered seedlings (LLWand HLW) were watered
twice a week to maintain a 20–25% soil moisture
content (close to field capacity). This protocol allowed
a long-term moderate water stress.
Twice during the experiment, four to five seedlings
per water regime � light combination were randomly
selected and one leaf per plant was harvested to
construct P–V curves following the methodology
proposed by Robichaux (1984). A short time re-
hydration, during not more than 3 h, was used to avoid
over-saturation of samples (Dreyer et al., 1990; Abrams
and Menges, 1992). In addition, predawn water
potential (Cpd) was measured to assess water stress
just before harvest.
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129120
3. Gas exchange
The same seedlings used for the construction of P–
V curves were employed to analyse the response in net
photosynthesis (A) to intercellular CO2 concentration
(Ci). Measurements were made in a climatic chamber,
at constant leaf temperature (23.4 � 0.7) and constant
water vapour pressure deficit (1.3 � 0.1 KPa), with an
open gas analyser working at differential mode (LCA-
4, Analytical Development Co., Hoddesdon, UK). For
a curve, each cork oak leaf was allowed to reach a
steady-state of 300 mmol mol�1 ambient CO2. This
concentration was reached after 20–30 min of
stabilization. Then, the concentration of CO2 into
the leaf chamber was lowered to 50 ppm by diverting
the air through a soda lime column and each new
concentration was set to a constant value by the IRGA
software. Following this schedule, several records
were taken between 300 and 50 mmol mol�1. Four to
six additional points were recorded between 300 and
1200 mmol mol�1 through increases of 200 mmol
mol�1 in each step. The 1200 mmol mol�1 concentra-
tion was enough to saturate photosynthesis in most
cases (Fig. 1). The CO2 concentrations above ambient
CO2 were achieved pumping free air and a CO2/N2
mixture through a bottle filled with water. During the
measurements, leaves were illuminated with 1200 and
1500 mmol m�2 s�1 PPFD, which were saturating
values for seedlings grown in the shade and sun
environments, respectively. Previously, it was proved
Fig. 1. A/Ci response in seedlings growing under high (HL—&, *)
and low light (LL—&, *) availability. Points are from four–five
seedlings in the first (circle) and second (square) harvest, respec-
tively. In subsequent analysis, parameters derived after adjusting the
model of Farquhar et al. (1980) to each curve were pooled in an
unique analysis as no differences were observed between harvests.
that both light levels did not bring about photoinhibition
during the construction of the curve. Light was provided
by an artificial light source. Data were treated with the
Photosyn Assistant version 1.1.2 software (Dundee
Scientific). Farquhar et al. (1980) model was fitted to the
empirical data using the equations of Harley et al. (1992)
to calculate the temperature dependent biochemical
parameters t, Kc and K0. Additionally, gas exchange in
water-stressed seedlings was assessed at an ambient
CO2 concentration of 365 mmol mol�1, under the same
irradiance and leaf temperature used for the construction
of A–Ci curves in well-watered plants. The difficulty to
maintain a steady-state for stomatal leaf conductance to
water vapour (gwv) during the construction of A–Ci
curves did not allow to estimate Vcmax and Jmax in water-
stressed seedlings. Reference values for A and gwv on
water-stressed and well-watered seedlings under ambi-
ent CO2 (365 ppm) were measured just after Cpd
measurements.
4. Nitrogen content and morphological
parameters
Leaves used for gas exchange measurements were
immediately harvested and leaf area estimated with an
image analyser (Delta-T devices, Hoddesdon, UK).
Leaf disks were taken to measure chlorophyll content
(Chlmax) according to Barnes et al. (1992). Leaf
samples were oven-dried at 80 8C. SLAwas calculated
as the ratio of fresh leaf area to dry weight. In these
same leaves, total leaf nitrogen content was analysed
by the Kjeldahl method.
A multifactor analysis of variance (ANOVA) was
applied to the data to compare the effects of light and
water stress on the different parameters. In a first
approach, the two harvests were analysed indepen-
dently. When no significant differences between
harvests were found, data were pooled for analysis.
A LSD multiple range test was used to test differences
between mean values (P < 0.05). Regression analysis
was made to show the change in gas exchange
parameters with water stress. The influence of light
availability on these responses was evaluated by
comparison of slopes and intercepts of the regressions.
Log-transformation of data was applied to compare
the relationship between A and gwv taking Cpd as
surrogate of water stress.
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129 121
5. Results
5.1. Drought imposition during the experiment
Soil moisture, integrated along the rooting zone,
was not significantly different in HL and LL plants
(P > 0.05), therefore the differences will be only
attributed to drought treatment. Well-watered plants
had an average soil moisture in the rooting zone of
25%, during most of the experiment. No interaction
between light and water was observed. Twelve weeks
from the beginning, available water in stressed plants
was maintained at around 50% the value in well-
watered seedlings, after integrating the three measure-
ments made along the rooting zone (total average soil
moisture 15%). Afterwards, water-stressed seedlings
were maintained with integrated average soil moisture
of 15% until the end of the experiment, just before the
second harvest.
Differences in water stress assessed by Cpd were
not significant between sun and shade seedlings,
independently of water regime (Fig. 2). Whatever
irradiance, the differences in Cpd were mainly related
Fig. 2. Predawn water potential (Cpd: MPa), osmotic potential at full (Cp
turgor lost point (RWC0: %) in cork oak seedlings growing under sun and s
pooling data from both harvests). W—well-watered and S—water-stresse
to watering level. Thus, the experimental protocol
used to irrigate seedlings allowed to maintain the same
water stress – in terms of average water availability –
during all the experiment, for seedlings grown under
both light environments.
5.2. Water relations
No differences between harvests were found for
any of the water-relations parameters, thus, the
analysis for pooled data was used.
There was a dependence of Cp100 and Cp0 on water
regime and relative irradiance (Table 1), although their
variation was mostly explained by water availability
(higher F value in the ANOVA analysis). Thus, in
both light treatments there was a decrease inCp100 and
Cp0 with drought (Fig. 2). This was confirmed by the
positive relationships between Cpd and Cp100 or Cp0,
which only showed significant differences in inter-
cepts related to light treatment, when a parallelism test
was done (Fig. 3). Despite the lower variance was
explained by the degree of light exposition, this factor
was also significant. In this sense, seedlings in the
100: MPa) and zero turgor (Cp0: MPa) and relative water content at
hade conditions submitted to a prolonged water stress cycle (n = 10,
d seedlings.
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129122
Table 1
Results of analysis of variance with watering and relative irradiance
during growth as sources of variation
Factor Variable d.f. F P
Relative irradiance Cp100 1 4.92 0.033
Cp0 1 5.90 0.021
CHR0 1 3.02 0.091
Watering Cp100 1 10.10 0.003
Cp0 1 33.26 0.000
CHR0 1 24.28 0.000
S � W Cp100 1 0.054 0.818
Cp0 1 0.336 0.566
CHR0 1 1.634 0.210
Fig. 4. Total chlorophyll (Clhmass) and the chlorophyll/nitrogen
ratio increased with SLA.
shade showed a trend towards a lower Cp0 and Cp100
at a same Cpd than those in the sun whatever water
stress. In both light treatments, CHR0 decreased in
response to water stress, with no significant differ-
ences between sun and shade seedlings (Fig. 2).
Fig. 3. Dependence of osmotic potentials at full (Cp100: upper
graph) and zero turgor (Cp0: lower graph) on predawn water
potential (Cpd). Intercepts for HL (black points and continuous
line) and LL (white points and dotted line) were significantly
different in the relationship between Cpd and Cp0. The linear
regression equation after adjustment for the same slope are HL:
Cp0 = �2.22 + 0.61Cpd and LL: Cp0 = �1.94 + 0.61Cpd, r2 = 0.42
(P < 0.001) and HL: Cp100 = �1.88 + 0.24Cpd and LL:
Cp100 = �1.72 + 0.24Cpd, r2 = 0.26 (P < 0.0047).
5.3. Specific leaf area, chlorophyll content and leaf
nitrogen content
Changes in SLA were only linked to the light
environment (P < 0.0001). Water stress did not
influence SLA in HL or LL seedlings (Table 2).
Watering did not change Chlmax since no differ-
ences between well-watered and water-stressed seed-
lings were observed (P > 0.05). Total chlorophyll
content on a mass basis as well as the inversion of
chlorophyll per unit of nitrogen (Chlmax/Nmass,
Table 2) were light-dependent. Both tended to increase
with SLA (Fig. 4), which on the other hand, was
explained mainly by the light environment.
Nmass was significantly higher in seedlings under low
light (Table 2). However, considering the differences in
SLA, a higher Narea was found under high light. Thus,
both watering as light explained Nmass changes. Nmass
was higher in water-stressed seedlings, increasing also
with shading.
5.4. Gas exchange
Well-watered and water-stressed seedlings showed
higher A and gwv under HL conditions. A and gwv
decreased in response to water stress in HL and LL
seedlings (Table 3). A negative relationship was found
between Cpd and A or gwv, under both light
environments (Fig. 5a and b). However, the relative
decrease in gwv with Cpd was more noticeable in
seedlings grown under high light. This was confirmed
by the significant interaction of light � watering in the
ANOVA analysis (P < 0.02). This result would point
out the higher sensitivity of gwv to water stress with
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129 123
Table 2
Different leaf structural and biochemical characteristics in seedlings growing under high and low light and submitted to two watering regimes
(n = 10; x � S:E:)
Treatment
HLW HLS LLW LLS
SLA (cm2 mg�1) 0.116 � 0.003 a 0.116 � 0.004 a 0.180 � 0.004 b 0.190 � 0.005 b
Nmass (mg g�1) 21.63 � 0.72 a 26.07 � 0.66 b 24.27 � 0.38 b 28.66 � 1.02 c
Narea (g m�2) 1.89 � 0.07 b 2.23 � 0.10 c 1.35 � 0.03 a 1.52 � 0.06 a
Chlmass (mg g�1) 10.79 � 0.48 a 11.94 � 0.71 a 16.03 � 0.56 b 16.40 � 1.12 b
Chl/N (mg g�1) 0.50 � 0.03 ab 0.46 � 0.03 a 0.66 � 0.03 c 0.57 � 0.05 bc
Data of the two harvests were pooled. SLA—specific leaf area, Nmass—nitrogen in a mass basis, Narea—nitrogen in an area basis, Chlmass—total
chlorophyll in a mass basis, Chlarea—total chlorophyll in an area basis, Chl/N ratio. HLW (high light–well-watered), HLS (high light–stressed),
LLW (low light–well-watered) and LLS (low light–stressed).
increasing irradiance. When Cpd decreased, gwv
decreased proportionally more than A, and this
resulted in higher IWUE for water-stressed seedlings,
whatever light environment (Table 2). However,
besides the interaction watering � light, the effect
of light alone was also marginally significant. There
was a trend for a higher increase in IWUE for HL
Fig. 5. Relationship between net photosynthesis (a: A,
mmol m�2 s�1) or stomatal conductance to water vapour (b: gwv,
mmol m�2 s�1) and predawn water potential as surrogate of water
stress degree (Cpd: MPa). Measurements on well-watered and water-
stressed seedlings were considered together in the same trend. The
relationships were different according to the light treatment when
data were log-transformed and the slopes compared.
seedlings under water stress (P = 0.072 and 0.088 for
light treatment and light � watering interaction
respectively, from ANOVA). The relationship between
Cpd and IWUE was only significant for HL seedlings
(Fig. 6), which reinforces the aforementioned argu-
ments about a steeper increase of IWUE in HL
seedlings under water stress. In a similar way,
seedlings from HL showed a higher A for the same
gwv when data of stressed and well-watered plants
were pooled (Fig. 7).
5.5. Photosynthetic parameters derived from
A–Ci curves
Net photosynthesis at ambient CO2 (Aamb) tended
to saturate in sun- and shade-leaves at Ci values around
40 to 30 Pa, respectively (Fig. 1). HL seedlings
showed higher Vcmax and Jmax estimated from data in
Fig. 1 (Table 3). The increase in Vcmax and Jmax ran in
Fig. 6. Response of intrinsic water use efficiency (IWUE: mmol -
mol) to predawn water potential (Cpd: MPa). The relationship was
only significant in seedlings grown under high light (black points).
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129124
Table 3
Different physiological parameters in seedlings growing under high and low light and submitted to two watering regimes (n = 10; x � S:E:)
Treatment
HLW HLS LLW LLS
Anet (mmol m�2 s�1) 11.65 � 0.68 a 7.21 � 0.91 b 7.15 � 0.40 b 4.15 � 0.67 c
gwv (mmol m�2 s�1) 167 � 11 a 70 � 12 c 100 � 7 b 50 � 8 c
Ci/Ca 0.581 � 0.018 b 0.471 � 0.031 a 0.606 � 0.021 b 0.578 � 0.039 b
IWUE (mmol mol�1) 75 � 4 b 108 � 7 a 73 � 5 b 85 � 9 b
Vcmax (mmol m�2 s�1) 50.54 � 2.88 a * 27.04 � 1.59 b *
Jmax (mmol m�2 s�1) 136.7 � 10.40 a * 68.73 � 3.62 b *
Fv/Fm 0.800 � 0.004 a 0.779 � 0.018 ab 0.799 � 0.007 a 0.765 � 0.011 b
Data of the two harvests were pooled. Anet—net photosynthesis, gwv—stomatal conductance to water vapour, Ci/Ca—ratio of CO2 mole fraction
at evaporating surface vs. ambient air, IWUE—intrinsic water use efficiency, Vcmax—maximal rate of carboxylation, Jmax—maximal rate of
electron transport, Fv/Fm—variable to maximum fluorescence ratio. HLW (high light–well-watered), HLS (high light–stressed), LLW (low
light–well-watered) and LLS (low light–stressed).
Fig. 7. Relationship between net photosynthesis (A, mmol m�2 s�1)
and stomatal conductance to water vapour (gwv, mmol m�2 s�1) in
seedlings growing so much in high or low light environments.
Intercepts were significantly different.
Fig. 8. Maximum rate of carboxylation (Vcmax) and light-saturated
rate of electron transport (Jmax) were highly correlated. A unique
relationship was established for well-watered HL and LL seedlings.
Each point resulted from adjusting the Farquhar et al. (1980) model
to the data in Fig. 1.
parallel (Fig. 8), so the quotient between carboxyla-
tion and electronic transport capacity was relatively
constant in both treatments (data not shown). In
addition, there was a significant negative relationship
between Vcmax and Jmax with SLA (Fig. 9a). The ratios
Fig. 9. (a) Relationship between maximum rate of carboxylation
(Vcmax) and light-saturated rate of electron transport (Jmax) to
specific leaf area (SLA: cm2 g�1) on well-watered seedlings of cork
oak; (b) relationship between the rates of Vcmax and Jmax to nitrogen
content on a area basis (Vcmax/Narea; Jmax/Narea) and the SLA.
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129 125
of Vcmax and Jmax to Narea showed the same trend, and
decreased with SLA (Fig. 9b).
6. Discussion
6.1. Water relations
Low water availability and shade, may be the
bottleneck for recruitment and survival of seedlings in
the understory of mature stands in Mediterranean
areas. Trees show different responses to overcome
resource limitation (Kloeppel et al., 1993; Canham
et al., 1996; Pages et al., 2003), but an important
question to be answered is if a low level of light might
limit their ability to cope with low water availability.
Shade and water stress have been found significant
for the development of mechanisms of water stress
tolerance, such as osmotic adjustment, in cork oak
seedlings (Groom and Lamont, 1997). In our study, no
interaction was observed between light and water
availability, although Cp0 was lower under high light,
whatever water regime. This result was confirmed by a
slightly higher but significant, intercept in the
relationship between Cpd and p0 in shaded seedlings.
The lower Cp0 and Cp100 in sun-grown seedlings has
been previously reported, but the differential impact of
contrasted light environments, under the same low
water availability has been seldom considered (Auge
et al., 1990; Aranda et al., 2001; Delperee et al., 2003).
Thus, it may be hypothesized the additive effect of soil
dryness and high sunlight exposure as environmental
factors which would trigger different drought toler-
ance mechanisms, such as the decrease ofCp0 (Groom
and Lamont, 1997).
One shortcoming of the present study is the absence
of a water status measurement at midday, which would
be surely lower in sun-seedlings (Kloeppel et al.,
1993), although not necessarily (Tschaplinski et al.,
1998; Valladares and Pearcy, 2002). In any case, for
the same value of Cpd, the results showed a higher
water stress in terms of turgor at dawn for shade
seedlings – calculated from the difference between
Cpd andCp0 – as a consequence of a lower capacity for
osmoregulation. This fact stressed the importance
under drought conditions to develop mechanisms such
as osmotic adjustment, in terms of turgor main-
tenance, under drought whatever light environment
(Abrams, 1986; Auge et al., 1990; Kloeppel et al.,
1994).
6.2. Changes in SLA, chlorophyll and nitrogen
contents
The change in SLA was a function of light with no
effect of water stress. Irradiance is known to be the main
factor that promotes shifts in SLA (Ellsworth and Reich,
1992, 1993; Niinemets and Kull, 1994; Niinemets and
Tenhunen, 1997), although water stress has also been
reported as an environmental factor that may modify
SLA (Waring, 1991). The increase in leaf area per unit of
dry matter has been interpreted as a mechanism to
optimize light harvesting under low light environments.
However, the higher leaf area per volume derived from
higher SLA in shade-seedlings would be less efficient to
control water losses under drought conditions. The
higher weight of light over water stress in determining
this character, would imply a remarkable role of light in
the acclimation of seedlings to shade environments,
despite the increasing risk for a lower tolerance to
drought. Therefore, changes in SLA might be inter-
preted as a homeostatic mechanism which prioritized
the optimisation of light capture (Lambers and Poorter,
1992). This fact was confirmed in our study by the
highest investment of nitrogen in chlorophyll in shade-
seedlings. This has been frequently interpreted as a
higher nitrogen allocation for light harvesting, under
light-limited conditions (Seemann et al., 1987; Evans,
1989; Niinemets and Tenhunen, 1997; Evans and
Poorter, 2001).
The decrease on the Chl/N ratio in a similar way for
shade- and sun-seedlings subjected to water stress
reinforced the idea of a similar negative impact of
water stress on the harvesting of light. This negative
impact was supported by the Fv/ Fm value, which was
slightly lower in leaves of seedlings submitted to
drought, under both light environments. However, a
value of Fv/ Fm close to 0.8 (Table 3) which is
considered the reference value for healthy leaves
(Bjorkman and Demmig, 1987; Adams et al., 1990),
reinforced the idea that the stomatal control of water
loss plays a major role as a mechanism to limit net
photosynthesis, for the range of water stress experi-
enced by seedlings in our study.
Watering and shade altered nitrogen content on a
mass basis (Nmass). Thus, there was a trend for a lower
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129126
Nmass in HL leaves. Previous results report the same
Nmass in leaves developed under sun or shade
conditions (Ellsworth and Reich, 1993; Evans and
Poorter, 2001), but also an increase on Nmass with
shade, depending on the species (Ellsworth and Reich,
1992; Niinemets and Tenhunen, 1997). Unexpectedly,
water stress increased Nmass both in HL and LL
seedlings, this could be related to a decline in growth
(Llorens et al., 2003). Despite differences in Nmass
between HL and LL seedlings, larger investment in
mass per leaf area in HL seedlings rendered a higher
Narea. This result highlights the role of SLA as the
main determinant of nitrogen partitioning on a leaf
area basis (Rosati et al., 2000; Aranda et al., 2004).
The resultant was a high degree of coupling in well-
watered seedlings between Narea and Asat, Vcmax and
Jmax, as it has been frequently reported (Niinemets and
Kull, 1998; Niinemets et al., 1998; Frak et al., 2001).
6.3. Gas exchange
A general pattern of the physiological response to
drought under different shade conditions has not
been previously described, in terms of gas exchange.
Three possible responses have been pointed out in
the literature. First, a very negative impact on
drought tolerance under shade (Vance and Zaerr,
1991; Fisher et al., 1991); second, a less negative
effect for shade growing seedlings (Abrams et al.,
1992); and third, a similar role of drought on the
pattern of physiological response at both extremes of
the light environmental gradient (Ogren and Oquist,
1985; Muraoka et al., 2002). In the present study,
photosynthetic capacity measured at saturating
irradiance was lower in shade grown seedlings
(Kloeppel et al., 1993; Abrams and Mostoller, 1995;
Landhausser and Lieffers, 2001). In well-watered
seedlings Vcmax and Jmax increased with relative
irradiance (DeJong and Doyle, 1985; Niinemets and
Kull, 1998; Le Roux et al., 2001; Robakowski et al.,
2003), the improvement in maximum carboxylation
rate running in parallel to the increase in maximum
electronic transport capacity. Such increase was
mainly a consequence of changes in SLA, which
resulted in a higher investment of nitrogen per area
under high irradiance and a higher return in terms of
Vcmax and Jmax, as previously reported (Kull and
Niinemets, 1998; Frak et al., 2002).
The higher photosynthetic capacity of sun-grown
seedlings was also maintained in water-stressed
plants. These seedlings showed higher net carbon
assimilation, whatever water stress. This result was
linked mainly to a lower stomatal limitation to
carbon acquisition for high Cpd and to the higher
photosynthetic capacity. On the contrary, in a similar
study where the combined effects of shade and
drought were assessed, Holmgren (2000) observed a
decrease in gwv under water stress only in sun grown
plants. He concluded that rates of the carbon fixed in
photosynthesis decrease under dry conditions only
for plants grown in high light environments. In our
study, seedlings growing both under HL and LL
conditions underwent a decrease in gwv and A with
water stress (Fig. 5a and b). However, there was a
trend for a higher stomatal control of water loss in
sun-seedlings. Thus, IWUE increased 30%, as
consequence of water stress in sun-seedlings, while
in shade-seedlings the increase was only of 12.24%.
Despite the differences between light treatments, as
well as to the fact that the interaction between light
and water regime was marginally significant, HL
seedlings tended to be more efficient in water use.
Indeed, that was reinforced by the relationship
between IWUE and Cpd, which was only significant
for HL seedlings.
Photosynthesis decreased in a similar fashion with
water stress, under both light environments, but the
decrease was not so steep as for gwv. As a consequence,
gwv had a higher weight as determinant of the increase
in IWUE under water stress. A positive relationship
has been showed between water use efficiency
assessed by isotopic discrimination and relative
irradiance (Jackson et al., 1993), but there is little
information about the interaction of light and water
availability (Groom and Lamont, 1997; Bonal et al.,
2000). The larger increase of IWUE in HL seedlings
with drought relied on a differential decrease in gwv
with water stress, which was proportionally higher for
HL plants, in the range of �0.2 to �1.0 MPa (Fig. 5a).
Thus, the higher sensitivity, in terms of stomatal
closure, in HL seedlings during the first stages of
drought, will allow them to respond earlier and more
efficiently to such stress in comparison to shade-
acclimated seedlings.
In conclusion, the less efficiency to develop
physiological mechanisms of water stress tolerance
I. Aranda et al. / Forest Ecology and Management 210 (2005) 117–129 127
in the shade, such as osmotic adjustment, or an
effective control of water loss, might put under risk the
long-term survival in the of seedlings growing under a
very low light and limitant water availability (Vance
and Zaerr, 1991; Delperee et al., 2003). However, it
should be recognized that other morphological
mechanisms focused to minimize the demand for
both requirements may play a more important role in
the adaptation to environmental conditions of drought
interacting with shade (Sack and Grubb, 2002; Sack
et al., 2003). The water stress applied in this study may
be considerate as moderate. Thus, a higher intensity of
drought, given by lower Cpd, might increase the
differences found in the range of water availabilities
considered. On the other hand, the responses reported
at present may be related to a threshold irradiance;
thus, in the future it would be interesting to assess the
water stress � light interaction in a wider range of
PPFD and water availability.
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