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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withdrawn after germination and seedlings were allowed to dry to a water content of ca. 50% of field capacity.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: aranda@inia.es (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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