Introduction 

Quercus suber L. is an evergreen oak growing across a wide range of environmental conditions in the Mediterranean Basin. Due to these diverse conditions, Mediterranean forest tree species are exposed to a range of selective pressures that may lead to phenotypic plasticity and local adaptation ([30]). Light is one of the most heterogeneous factors affecting plant growth and development ([7]) and acclimation of leaf photosynthesis to different light environments is a key factor that maximizes carbon gain ([8], [18]). Acclimation to fluctuations in light conditions plays a crucial role in determining the competitive ability of forest tree species ([14]). Acclimation to environmental stresses is achieved through structural, morphological and physiological adjustments at both leaf and whole-plant level ([27], [33], [14]). The acclimation of plants to high light condition appeared in the ability to use high photosynthetic photon flux densities efficiently ([14], [35]).

Plants can form sun and shade leaves in response to the variation in growth irradiance. Sun leaves have a higher photosynthetic capacity than shade leaves. Higher photosynthetic capacities in sun leaves correlate with a greater leaf thickness and is supported by a greater investment of nitrogen in photosynthetic enzymes ([4], [9], [28], [37], [8], [23], [24], [25]). Furthermore, strong correlations were reported between photosynthetic capacity and surface area of mesophyll cells ([25]), between photosynthetic capacity and the chloroplast area facing the intercellular space ([23]) and between photosynthetic capacity and stomatal and mesophyll conductances to CO₂ diffusion ([16]). Pandey & Kushwaha ([25]) studied the relationship of leaf anatomy with photosynthetic acclimation in Valeriana jatamansi under full irradiance [FI, 1600 μmol (PPFD) m^-2 s^-1] and shade conditions [SC, 650 μmol (PPFD) m^-2 s^-1]. FI plants had thicker leaves with lower specific leaf area (SLA), enabling to arrange all their chloroplasts along the mesophyll cell surface. Leaf thickness is determined by the irradiance during leaf development, and it changes little after leaf maturation ([32]). Nevertheless, when leaves are subjected to higher irradiance after maturation, their photosynthetic capacity often increases. However, high photosynthetic capacity is not always associated with thicker leaves.

The aim of this study was to test whether the habitat of origin induced an intraspecific variation in response to transfer of plants from low to high growth irradiance. To this purpose, seedlings of cork oak (Quercus suber L.) originating from two different natural habitats and representing a marked climate gradient were used. Changes in photosynthetic characteristics of mature leaves were examined when transferred from low to full sunlight condition. The photosynthetic capacity, the maximum carboxylation rate of Rubisco (Vc_max), the potential light-saturated electron transport rate (J_max) and the specific leaf area were determined for leaves grown under different light conditions.

  Materials and methods 

Seed origin and experimental design

Acorns of two Quercus suber populations originating from contrasting environments in the northwestern provinces of Tunisia were collected in October 2010. The first site, the National Park of Feija (36° 30′ 00″ N, 08° 20′ 00″ E), is located in the North of the Kroumirie Mountains and is characterized by a cold and humid climate. The altitude varies between 800 and 1500 m a.s.l. and the average annual rainfall is 1217 mm, with precipitation increasing with altitude and reaching 1800 mm year^-1. In January, the average temperature is 7 °C and can drop to 0 °C with snow which falls every year. In summer, the temperature rises notably and averages around 29 °C. This leads to a mean annual temperature of 14.3 °C. Cork oak trees are found in forest mosaics along with other tree species, including Zeen oaks (Quercus faginea), maritime pines (Pinus pinaster) and many shrub species.

The second site is located in Gaâfour (36° 32′ 190″ N, 09° 32′ 40″ E) in the southern hills and plains around the city of Siliana. It is characterized by a semi-arid climate (480 mm year^-1) with moderate winters and hot dry summers. Cork oak trees at this site are found in agroforestry systems containing olive trees. The altitude is 560 m a.s.l. and the mean annual temperature is 17 °C.

Immediately after collection, acorns were planted in a common greenhouse at the National Research Institute for Rural Engineering, Waters, and Forestry under low light conditions (LL, 15% of full sunlight). On October 1^st, 2013, low-light adapted seedlings were growing in 5 litres pots containing a mixture of equal proportions of soil and compost. Seedlings were randomly assigned to one of two light treatments for 40 days: (i) high light (100% natural incident irradiance, HL); and (ii) low light (LL). In the LL treatments, light levels were obtained through the use of layers of neutral shade-cloth, while in the HL treatment seedlings were left uncovered. On a sunny day, photosynthetically active radiation (PAR), measured using a Li-190^® device (Li-Cor Bioscience, Lincoln, NE, USA) ranged between 1630-1810 μmol m^-2 s^-1 and 215-315 μmol m^-2 s^-1 in the HL and LL treatments, respectively. Irrigation to saturation was provided manually each day. Mean volumetric soil water contents of the pots containing seedlings in the two light treatments was monitored by a time domain refractometry (TDR, Trase system I, Soil moisture Equipment Corp., USA) and was approximately 25-30%. To ensure that all seedlings were exposed to a similar range of light throughout the experiment and minimize the possible effects of within-block light variability, each block containing twenty pots (ten for each provenance per light treatment) was periodically moved. All experiments were carried out using mature leaves that were fully expanded and developed prior to the light treatment.

CO₂ response curves

Photosynthetic traits were measured in situ on mature leaves of four to six different seedlings per treatment (one leaf per plant) during the late morning (08:00-11:00 h) and early afternoon (13:00 -16:00 h). At the beginning of the experiment, leaf gas exchange was measured in three low light-adapted seedlings from each provenance. After 40 days, four plants from Gaâfour and Feija provenances were transferred from low to high light and used to carry out gas exchange measurements. Three additional LL adapted Gaâfour and Feija seedlings were used to ensure that there was no change of photosynthesis parameters during the experiment for plants kept under LL. Photosynthetic carbon dioxide response curves were recorded using a portable gas-exchange system (LI-6400^®, Li-Cor Inc., Lincoln, NE, USA) equipped with a 2 × 3 cm light-source chamber (6400-02B LED^®, Li-Cor). Each leaf was adapted to dark for approximately 30 minutes in the measurement chamber. When the gas exchange reached a steady state, net CO₂ efflux was recorded as an estimate of dark respiration (Rd). At this point, incident PFD was set to 1600 µmol m^-2 s^-1 (saturating light). The leaf temperature was maintained at 25 °C and the humidity of the incoming air was kept at 50-60%. The external CO₂ partial pressure (C_a) variation consisted of 12 steps, starting by inducing photosynthesis at ambient CO₂ concentration of 400 µmol mol^-1 until net photosynthesis (A_n) stabilized (A_n varied by less than ± 2%). This was important to ensure a steady-state activation of Rubisco ([19]). The external CO₂ concentration (C_a) values were then decreased to 300, 200, 100 and 50 µmol CO₂ mol^-1 respectively. Upon completion of the measurement at 50 µmol CO₂ mol^-1, C_a was increased back to 400 ppm to check whether the original A_n could be restored. If this was achieved, C_a was increased stepwise to 600, 700, 1000, 1200, 1500 and 2000 ppm. Leaves were allowed to equilibrate for at least 5 minutes at each step before data logging. At the end of each A-C_i curve, the leaf area and mass enclosed in the chamber was measured with a leaf area meter portable laser (Model Cl-202). The specific leaf area (SLA) was determined as the ratio of the leaf area to leaf dry mass of individual leaves. To estimate the CO₂-saturated rate of photosynthesis (A_sat), CO₂ response curves were fitted using a three components exponential function ([39]) according to the following equation (eqn. 1):

where A_n is leaf net photosynthetic rate and x is C_i. Using this equation, A_sat was calculated as (a+c). The CO₂ compensation point (Γ) was estimated from x-axis intercepts.

Estimation of V_cmax, J_max and relative stomatal limitation

Non-linear regression techniques, based on the equations of Farquhar et al. ([11]) and later modified by Sharkey ([31]) and Harley & Sharkey ([17]), were used to estimate V_cmax (the maximum rate of carboxylation limited by Rubisco) and J_max (the maximum rate of carboxylation limited by electron transport). In some cases, carboxylation may also be limited by triose phosphate availability ([31], [17]); however, this was not observed in any of the seedlings used in this study. V_cmax and J_max describe the upper limit to biochemical processes (amount, activity, and kinetics of Rubisco and regeneration of RuBP), both parameters are derived from different portions of the A_n-C_i curve. Points in the lower portion (at low Ci) of the curve are used for V_cmax and points in the upper portion (at high C_i) for J_max. The Michaelis-Menten constants for CO₂ and O₂ (K_c and K_o respectively) and the CO₂ compensation point in the absence of mitochondrial respiration (Γ*), as well as their temperatures dependencies, were taken from Bernacchi et al. ([3]).

The stomatal limitation was calculated according to Farquhar & Sharkey ([10]) as follows (eqn. 2):

where A_o represents carbon assimilation under natural ambient CO₂ in the absence of stomatal limitation.

Statistical analysis

Gas exchange measurements were repeated 4 and 6 times for HL and LL seedlings respectively. Mean values and standard errors were calculated. One-way analysis of variance (ANOVA) followed by the post-hoc Duncan’s test (α =0.05) were applied to test for differences among means of photosynthetic gas exchange parameters and specific leaf area.

Two-way analysis of variance was also conducted to test the interaction effect (provenance × light) on photosynthetic parameters. Pearson’s correlation between the photosynthetic parameters and SLA were also calculated. All ANOVA analyses were performed using the software package SigmaPlot^® (Systat Software Inc., San Jose, CA, USA). Pearson’s correlation analysis was done using XLSTAT v. 2014 (⇒ http:/­/­www.­xlstat.­com) after data standardization.

  Results 

Photosynthetic responses to transfer from low to high light

Gas exchange parameters between LL and HL leaves in Gaâfour and Feija seedlings at an ambient CO₂ concentration of 400 ppm were compared (Fig. 1). Stomatal conductance (g_s) and net carbon assimilation (A_n) measured under saturating light in LL leaves were found to be similar in both Feija and Gaâfour provenances.

Fig. 1 - Leaf physiological traits of leaves maintained continuously in low light (black) or transferred from low to high light (white): net photosynthesis (A_n, µmol m^-2 s^-1); dark respiration (Rd, µmol m^-2 s^-1); stomatal conductance (g_s, mmol m^-2 s^-1); intercellular CO₂ concentration (C_i, µmol mol^-1); relative stomatal limitation (L_s, %); and specific leaf area (SLA, cm² g^-1). Different uppercase letters indicate a significant difference between provenances in the same light environment, whereas different lowercase letters indicate significant difference between light environments in the same provenance (P ≤ 0.05).

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In HL leaves from the Gaâfour provenance, a significant increase in A_n was observed when compared to LL leaves (P < 0.01). While the g_s in this environment also increased, it was not significant (P > 0.05). No notable variations in these parameters (A_n and g_s) were detected in Feija leaves under different light condition treatments. At HL, A_n and g_s were significantly higher in Gaâfour leaves than in Feija leaves (P = 0.027 and 0.003 for A_n and g_s, respectively).

It was expected that the variations of g_s may explain significant differences of A_n in HL leaves of the two provenances. However, g_s did not show such differences, since stomatal limitation to carbon assimilation (L_s) remained similar in both provenances at HL conditions. Similarly, the intercellular CO₂ concentration (C_i) did not change in the HL leaves of Gaâfour and Feija seedlings when compared with LL leaves. A significant contribution of g_s to the lower A_n in Feija leaves at HL is therefore not supported. Although g_s did not differ between light treatments in the same provenance, there was a highly significant treatment × provenance interaction effect on both g_s and A_n (P < 0.01 - Tab. 1).

There were no significant changes to the leaf dark respiration in the seedlings of Feija transferred from LL to HL in comparison to those maintained at LL (P > 0.05). Dark respiration was highest in HL leaves of Gaâfour provenance (Tab. 1) but did not show a significant treatment × provenance interaction effect (Tab. 1). No significant changes in specific leaf area were measured between leaves of seedlings maintained at LL and those transferred to a HL condition in either provenance (Fig. 1).

Photosynthesis in response to C_i

In order to characterize the photosynthetic activity in the leaves of Gaâfour and Feija provenances under the two light conditions, A_n was measured as a function of the intercellular CO₂ concentration (C_i). As shown in Fig. 2 (insert), A_n of LL leaves in Gaâfour provenance was similar to Feija provenance at low C_i but it was higher at high C_i. In the HL leaves of Gaâfour, A_n as a function of C_i increased in comparison to LL leaves. No differences were observed between Feija seedlings grown in either light condition.

Fig. 2 - Net photosynthesis (A_n) as a function of intercellular CO₂ concentration (C_i) of leaves maintained continuously in low light (solid line) or transferred from low to high light (dotted line) in Feija and Gaâfour provenances. Inserts: Feija (closed symbols) and Gaâfour (open symbols).

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Fig. 3 shows that the CO₂ compensation point (Γ) remained constant in leaves of both provenances when the leaves were transferred to full sunlight, despite a negligible decrease detected in Gaâfour leaves. Under both light regimes, leaves of Gaâfour seedlings had significantly higher CO₂-saturated photosynthesis rates (A_sat) than those of Feija (P < 0.05). The statistical analysis (two-way ANOVA), did not show any significant treatment × provenance interaction effect on either Γ or A_sat (Tab. 1).

Fig. 3 - Photosynthetic parameters estimated from A_n/C_i curves of leaves maintained continuously in low light (black) or transferred from low to high light (white): the CO₂-saturated net photosynthesis (µmol CO₂ m^-2 s^-1), the CO₂ compensation point Γ (ppm), the maximum ribulose 1.5- bisphosphate carboxylase/oxygenase (Rubisco) carboxylation V_cmax (µmol CO₂ m^-2 s^-1) and the potential light-saturated electron transport rate J_max (µmol m^-2 s^-1). Different uppercase letters indicate a significant difference between provenances in the same light environment, whereas different lowercase letters indicate significant difference between light environments in the same provenance (P ≤ 0.05).

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Estimation of V_cmax and J_max

Stomatal constraints on photosynthesis were similar in the Feija and Gaâfour provenances, indicating that photosynthetic acclimation was curtailed at the biochemical level. The parameters describing the maximum rate of carboxylation by Rubisco (V_cmax) and the maximum rate of electron transport (J_max) are shown in Fig. 3. In the LL leaves, V_cmax was higher in Feija than Gaâfour leaves (P< 0.05). When transferred to HL conditions, V_cmax increased significantly in the Gaâfour seedlings only. This increase of V_cmax in HL leaves of Gaâfour showed approximately a 60% enhancement compared to leaves that remained in LL conditions.

Although J_max in LL leaves were similar in Feija and Gaâfour provenances, Feija seedlings displayed significantly lower J_max after 40 days of growth under HL conditions. This suggests a lower capacity of electron transport in Feija leaves, leading to the limitation of RuBP regeneration. The statistical analysis showed a highly significant treatment × provenance interaction effect on V_cmax, but this interaction was non-significant on J_max (Tab. 1).

  Discussion 

We studied the effect of increasing light intensity on photosynthesis in cork oak (Quercus suber L.) seedlings of two provenances differing in climatic conditions at their geographical origin. Three-year-old cork oak seedlings of both provenances, grown in pots in a common greenhouse, were randomly assigned to one of two light treatments (HL, full sunlight and LL, 15% of sunlight).

The HL leaves of Gaâfour provenance proved to have a significantly higher photosynthetic capacity (P < 0.05) when compared to the Feija leaves. Different studies showed a possible intraspecific variability and phenotypic plasticity in Cork oak as an adaptation to contrasting regional climate conditions ([2], [1], [34], [30], [21], [15]). Investigating the influence of full-sun on seedling hardiness, Calzavara et al. ([6]) suggested that the acclimation process can induce changes in physiological, anatomical, and morphological traits of plants, favoring their establishment after transplantation to the field. The results of the present study indicate that the lower A_n in the HL leaves of Feija were not related to the limited stomatal conductance. Furthermore, the higher A_n in Gaâfour leaves at full sunlight as compared to those of Feija, was consistently observed at all C_i values applied (Fig. 2). These findings do not exclude that diffusional limitations are important factors affecting photosynthesis. However, it does highlight that additional, non-diffusive limitations affect photosynthesis in Feija leaves compared to those of Gaâfour for seedlings transferred to HL condition. It is widely assumed that J_max and V_cmax parameters represent the major limitations to light-saturated photosynthesis ([19]) and that the decrease in biochemical capacity (J_max and V_cmax) can limit A_n at high light, possibly by affecting nitrogen partitioning to Rubisco with increasing relative irradiance ([14], [18], [12]). Mechanistic photosynthetic models may be used to determine the impact of varying environmental conditions - including those predicted to be affected by climate change - on the biochemistry of photosynthesis and carbon acquisition at the leaf and plant levels ([26]). The model by Farquhar et al. ([11]) describes limitation processes in photosynthesis.

In this study V_cmax and J_max estimated from A_n/C_i curves were within the range observed for other Mediterranean species ([13], [1], [22], [38]). Correlations between the photosyntheticgas exchange parameters and SLA are shown in Tab. 2. The Rd, g_s, V_cmax and J_max were significantly and positively correlated with the A_n. The SLA and other gas exchange parameters were not significantly correlated with the A_n. This correlation analysis elucidates that V_cmax and J_max are the major contributors in the photosynthetic acclimation to HL conditions in cork oak.

The results from this study indicate that only Gaâfour seedlings were able to acclimate physiologically to a high light environment. This is in accordance with previous results indicating that the increase in V_cmax and J_max reflects a physiological plasticity at the cell levels caused by an increase in the photosynthetic capacity per unit of leaf tissue ([14]). The high photosynthetic capacity of HL leaves of Gaâfour was accompanied by a significant increase (P < 0.05) of the dark respiration.

A previous study showed that a large investment of nitrogen in photosynthetic enzymes (especially Rubisco) supports the increase of photosynthetic capacity in spinach leaves transferred from low to high growth irradiance ([36]). It also increases the respiration rate in wheat leaves ([20]). [23] suggest that this increase in respiration rate in Chenopodium album is related to the maintenance processes of leaves, e.g., protein turnover which consumes respiratory energy. The high dark respiration in HL leaves confirms the acclimation ability of Gaâfour seedlings to full sunlight, since variations in respiration have been widely proposed as a component of acclimation to photon availability ([25]). Similar to A_n, the Rd was significantly and positively correlated to g_s, V_cmax and J_max.

A remarkable finding of the experiment was that the SLA did not differ among provenances or light treatments (Fig. 1), suggesting that leaf function can acclimate to changing light conditions despite no structural or morphological adjustments. A very common morphological response to high light is a lower SLA (or high leaf mass area), which is generally due to increased leaf thickness ([5]). However, high photosynthetic capacity is not always accompanied by thicker leaves ([23]). Oguchi et al. ([23], [24]) explained the mechanism by showing that the transfer from low to high light increases the area of chloroplasts facing the intercellular space. The mesophyll cells of LL leaves had opened spaces along cell walls where chloroplasts were absent, which enabled the leaves to increase the photosynthetic maximum rate when they were exposed to high light. Leaf thickness determines an upper limit to the photosynthetic maximum rate of leaves subjected to a change from low to high light conditions. Shade leaves would only increase the photosynthetic maximum rate when they have open space to accommodate chloroplasts which elongate after light conditions improve, which possibly was not the case in Feija leaves. No significant correlation was observed between SLA and the gas exchange parameters (Tab. 2).

In conclusion, this finding corroborates previous results ([1], [29]) showing a large provenance-level differentiation in cork oak with provenance from dry places exhibiting the higher tolerance.

  List of abbreviations 

A_sat: CO₂-saturated photosynthesis; HL: high light; LL: low light; PAR: photosynthetically active radiation; A_n: net photosynthesis; PPFD: photosynthetic photon flux density; LED: light emitting diode; C_a and C_i: external and intercellular CO₂ molar fractions; g_s: stomatal conductance; Γ: CO₂ compensation point; L_s: stomatal limitation; J_max: maximum electron transport rate; V_cmax: ribulose 1-5 biphosphate carboxylation.

  Acknowledgments 

This work was supported by the National Research Institute for Rural Engineering, Waters, and Forestry in Tunis, Tunisia. We wish to thank Dr. Peter Streb (UniversitéParis-Sud 11, Ecologie, Systematique et Evolution, UMR-CNRS 8079, Orsay, France) for carefully reading the manuscript. We sincerely thank Mr. Ahmed Al-Fatlawi for his review of the English language of this manuscript.

  References