Free Access
Issue
Ann. For. Sci.
Volume 67, Number 3, May 2010
Article Number 308
Number of page(s) 10
Section Original articles
DOI https://doi.org/10.1051/forest/2009114
Published online 18 February 2010

© INRA, EDP Sciences, 2010

1. INTRODUCTION

Holm-oak (Quercus ilex L.) is a deep-rooted, evergreen dominant species in Mediterranean forests which has a great capacity for resprouting after fire, clear-cut, grazing or other disturbances. Resprouts after any of these events show decreased shoot/root ratios, which makes more water and nutrients available to the shoot than in the original plants and favours photosynthesis stimulation and rapid growth (Fleck et al., 1998). Q. ilex is exposed to multiple environmental stress factors such as drought, heat shock, chilling, nutrient deprivation and high light stress amongst others. Increased probability of drought, heat and rising atmospheric CO2 concentration during the coming decades may be particularly important in the Mediterranean basin (Christensen et al., 2007). Moreover, the expected increased risk of uncontrolled fire episodes could lead to the exhaustion of several species, generating a decline in their resprouting capacity and recovery.

There has been a long-standing controversy as to whether drought limits photosynthesis by stomatal closure, metabolic impairment or through diffusive resistances (Lawlor and Tezara, 2009). Of these resistances, CO2 transfer conductance inside the leaf or mesophyll conductance (gmes) is considered relevant to photosynthesis (Flexas et al., 2008). Metabolic photosynthesis limitations (e.g. injuries to photosynthetic biochemistry and photochemistry) during drought may only be apparent: drought produces low gs, closely related to gmes, resulting in a decreased availability of CO2 in the chloroplast, which down-regulates the biochemical machinery of photosynthesis. gmes can be affected by leaf morphology (Terashima et al., 2001); in fact, previous results of our group (Peña-Rojas et al., 2005) related changes in gmes in nursery-grown holm-oak plants submitted to water stress to variations in leaf anatomy and gas-exchange parameters.

Carbon isotope discrimination (Δ13C) is largely due to Rubisco (which discriminates against 13C during RuBP carboxylation), with the amount of discrimination depending on the ratio of CO2 partial pressure at the carboxylation site (CC) to CO2 partial pressure in the surrounding air (Ca), which is affected by gs and gmes (Farquhar et al., 1989). As described above, morphological characteristics can affect internal resistances; thus, leaf thickness and leaf density as components of the leaf mass per area parameter (LMA) (Niinemets, 1999), can be an important source of variation in Δ13C.

The aim of this study was to characterize the photosynthetic limitants during holm oak regrowth after a clear-cut, and especially the contribution of mesophyll conductance (gmes) under drought conditions. Two kinds of resprout were used for this study, which differed in their cutting season: winter, when plants had a high availability of stored underground reserves, and summer, when part of the stored reserves had already been remobilized and used to support early growth. Other aims were to relate the morphological characteristics of resprouts to the observed gmes and to examine the effect of gmes on carbon isotope discrimination (Δ13C) values. The characterization of the photosynthetic and growth limitations during Q. ilex resprouting after disturbances would help us to establish the adaptation capacity of this plant in the context of global change and biodiversity conservation in Mediterranean forests.

Table I

Climatological data recorded at the forest site during the gas-exchange measurements of the different treatments (control, C; winter resprouts, RW; summer resprouts, RS); data are the mean ±SE of all measurements.

2. MATERIALS AND METHODS

2.1. Experimental site and plant material

The study was carried out at Can Coll, Serra de Collserola forest, Barcelona, Spain; 41° 28′ 28′′ N, 2° 7′ 32′′ E. A plot (400 × 280 m) at altitude of 140 m and oriented N-NE was selected. The climate is Mediterranean, with cold winters, cool and wet springs and autumns, and hot dry summers (Tab. I). The 35-year old forest is dominated by Quercus ilex and Pinus halepensis. In February, 25 Quercus ilex plants were selected (5.9 ± 0.3 cm mean diameter at breast height (DBH), 4.7 ± 0.2 m mean height, 1.4 ± 0.2 kg mean leaf biomass) and the shoots of 10 randomly selected plants were completely excised 15 cm above soil level. Resprouts (R) after this date were designated as RW (winter resprouts). In August, 10 more plants were completely excised and resprouts after this were designated as RS (summer resprouts). Five plants were kept undisturbed, as controls (C) of the clear-cut site. Leaf gas exchange and chlorophyll fluorescence were measured in fully expanded leaves of the same age: in the first winter (W; February–March), only controls and RW leaves were analysed, as RS had resprouted badly in the autumn; in the subsequent summer (S; July–August), all treatments could be analysed. Samples were collected for 13C composition (δ13C), leaf mass per area (LMA), leaf density (D) and leaf thickness (T) determinations.

2.2. Leaf gas exchange

A portable gas exchange system LI-6200 (Li-Cor Inc., Lincoln, NE, USA) was used for punctual measurements at midday on nine attached, fully expanded, current-year leaves per treatment, season and leaf orientation. Leaf cuvette conditions differed according to the season (Tab. I). Results were expressed per leaf-projected area (LA), obtained with an Epson GT5000 scanner and processed using image analyser software. In each season, ten CO2 response curves of CO2 assimilation vs. intercellular CO2 concentration (A / Ci) were obtained per treatment on attached leaves with a LI-6400 instrument (Li-COR, Lincoln, Nebraska, USA). Leaf cuvette conditions were established according to the season and time of the day to reproduce a typical day in every season.

For A/Ci curves, PPFD was established as 600 μmolm−2 s−1, which is saturating under these conditions (Peña-Rojas et al., 2004); a range of ambient CO2 concentration (Ca) from 50 to 800 μmol mol−1 was covered. Analyses of the curves permitted the determination of: Amax, net photosynthesis at saturating Ci and PPFD; Vc,max, maximum carboxylation velocity of Rubisco; Jmax, maximum electron transport contributing to RuBP regeneration; ls, stomatal limitation to A(ls(%) = 100 × (1 − (A / Asat)); Asat, net photosynthesis at saturating light and Ci = 350 μmol mol−1.

To assess the effect of heterogeneous stomatal conductance across the leaf surface, steady-state chlorophyll fluorescence was measured in six spots of 27 leaves of the same plants used in the experiment. Water potential (Ψ) of the same leaves was also obtained with a Scholander-type pressure pump (Soil Moisture 3005, Soilmoisture Equipment Corp., Goleta, CA, USA). The coefficient of variation of ΦPSII (see below) was not statistically higher than system repetitiveness (around 9%), indicating the absence of patchiness, and did not correlate with Ψ.

2.3. Chlorophyll fluorescence and calculation of mesophyll conductance and CO2 concentration in the chloroplast

Chlorophyll fluorescence parameters were quantified with a portable modulated fluorometer (Mini-PAM Photosynthesis Yield Analyzer, Walz, Effeltrich, Germany) on the same leaves used for gas-exchange measurements. Fluorescence parameters (Fm, , Fo and Fv), photochemical PSII efficiency (ΦPSII) and the maximum quantum yield at midday (Fv / Fm) were determined as described (Fleck et al., 1998). Non-photochemical quenching (NPQ) was calculated using the Stern-Volmer equation: NPQ = . Adaptation took at least 20 min, after which Fv / Fm values reached about 95% of the pre-dawn values in Q. ilex (Fleck et al., 1998).

Mesophyll conductance (gmes) and CO2 concentration in the chloroplast (CC) were calculated from combined gas-exchange (LiCor 6400) and chlorophyll fluorescence (Mini-PAM) measurements, as described by Epron et al. (1995), and Galmés et al. (2007), except for respiration, which was calculated in the same leaves at the end of an A/PPFD curve after a five min acclimatisation to darkness. Galmés et al. (2007) showed that this method yields equivalent results to the “constant J”method (Harley et al., 1992), which makes no a priori assumption about the relationship between electron transport and fluorescence. Moreover, Flexas et al. (2007) demonstrated that both methods gave results that were comparable to Ethier and Livingston’s findings (2004), which did not rely on fluorescence measurements and to calculations by carbon isotope discrimination.

Table II

Midday values of net photosynthesis (A), stomatal conductance (gs), intercellular CO2 concentration (Ci), instantaneous water use efficiency (WUEi = A / gs), relative water content (RWC), PSII efficiency (ΦPSII), non-photochemical quenching of fluorescence (NPQ) and potential quantum yield of PSII at midday (Fv / Fm). Data are presented according to treatment (control, C; winter resprouts, RW; summer resprouts, RS), leaf orientation (north, south) and season (winter, summer). Values are mean ±SE of nine replicates. Significant differences across rows or columns (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and leaf orientation (α,β)).

The rate of electron transport (ETR) was calculated as ETR = ΦPSII × PPDF × 0.5 × 0.82, where 0.5 is a factor that assumes equal distribution of energy between the two photosystems and 0.82 is the light absorptance we obtained on Q. ilex leaves using an integrating sphere. According to the model of Epron et al. (1995), ETR can be divided into two component fractions, ETRA + ETRp, used for CO2 assimilation and for photorespiration, respectively. To calculate CC, we used S = (ETRA / ETRP) / (CC / O) (Laing et al., 1974), where S is the specificity factor of Rubisco and O is the oxygen model fraction in the air. We used a value of S = 93.3 mol mol−1 (Balaguer et al., 1996) that was corrected for leaf temperature according to Brooks and Farquhar (1985). The ratio between mesophyll conductance to CO2 and stomatal conductance (gmes / gs) was calculated at midday.

2.4. Leaf carbon isotope composition

Sixteen leaves per six plants per treatment and season were collected, oven-dried at 65 °C to constant dry weight and ground in a Mixer-Mill 8000 (Spex) in vials with tungsten carbide balls. Water-soluble extracts were prepared as follows: 2 g of dry material per plant were suspended in 25 mL water (3 replicates per plant) and were heated to 100 °C for 15 min; after cooling to room temperature, samples were filtered (Whatman nr. 1), stored at -40 °C and lyophilized. Approximately 4 mg of the lyophilized water-soluble extract (WSE) and 4 mg of dry mass (Md) were fed into a gas chromatograph (Carlo-Erba NA1500 Series II elemental analyser, CE Elantech, Inc., Lakewood, NJ, USA), connected on-line to an isotope ratio mass spectrometer (IRMS, Finnigan, Delta S; Thermo Finnigan, San Jose, CA, USA) for δ13C determination. δ13C values were determined using a standard calibrated against Pee Dee Belemnite (PDB) carbonate and used to estimate carbon isotope discrimination (Δ13C) as: Δ13C = 1000·(δaδp) / (1 + δp), where δa and δp are values for air (−7.8%0) and the plant, respectively (Farquhar et al., 1989).

Table III

Net CO2 assimilation at saturating Ci and light (Amax), maximum carboxylation velocity of Rubisco (Vc,max), maximum potential rate of electron transport contributing to RuBP regeneration (Jmax) and stomatal limitation (ls) from A/Ci curves for the different treatments (control, C; winter resprouts, RW; summer resprouts, RS) and season (winter, summer). In summer, the time of day was also considered. Each value represents the mean ±SE of ten replicates. Significant differences across rows or columns (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and time of the day (α,β,γ)).

2.5. Relative water content and leaf biomass parameters

Relative water content (RWC) was measured at midday in five young leaves of five plants per treatment. RWC was calculated as [(MfMd) / (MfsMd) × 100] , with Mf being plant fresh mass; MFS, plant fresh saturated mass (after rehydrating samples for 24 h in the dark); and MD, plant dry mass (after oven-drying samples at 65 °C until constant weight). Leaf mass per area, LMA, was determined (Md/LA), and its components (Mf/LA) and [(Md / Mf) × 100] , as indicators of leaf thickness (T) and leaf density (D), respectively (Niinemets, 1999), were calculated on the same plants as for gas-exchange measurements (30 leaves per treatment) in winter and summer.

2.6. Statistical analyses

All statistical procedures were carried out through the SPSS in Windows (v. 11.0, SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) tested the main effects and interactions, against appropriate error terms. Main factors per treatment and season for all variables were analysed. Leaf orientation was included for gas exchange and chlorophyll fluorescence analyses. The kinds of material analysed (WSE, DM) were included in the analyses of parameters derived from leaf δ13C. The post-hoc Duncan test was applied where suitable. Differences were considered significant at p ≤ 0.05. Only statistically significant differences are described in the Results and Discussion that follow.

3. RESULTS

Although the two kinds of resprout used for this study differed in their cutting season, the only difference found between them was the time the resprouts took to appear: RW resprouted in the following spring, 2–3 months after cutting, whereas RS resprouted badly in the autumn and were suitable for photosynthesis measurements only from the next spring onwards (7–8 months after cutting). Since from this moment on they showed no difference from RW plants in the parameters analysed, all kinds of resprouts will be considered as R in the Discussion section.

3.1. Leaf gas exchange and chlorophyll fluorescence

A, gs and instantaneous water use efficiency (WUEi = A / gs) at midday (Tab. II) showed no difference between treatments in winter, whereas in summer, resprouts gave higher values than C. Declines in A and gs between winter and summer were observed for all treatments, but were more pronounced in C, In all treatments ΦPSII and midday Fv / Fm values were lower in summer than in winter, whereas NPQ were lower in winter than in summer (Tab. II).

Data derived from the A/Ci curves performed under midday conditions (Tab. III) showed in winter no difference between R and C in Amax, Vc,max or Jmax. There was a decrease from winter to summer, with R showing higher values than C (Amax: 66.1%, Vc,max: 57.7%, Jmax: 59.3%, on average). Stomatal limitation (ls) was higher in C than in R in both seasons, with ls higher in summer than in winter for all treatments (52.1% higher on average). In summer, daily variations were observed for Amax with the highest values in the morning, whereas no difference was found in Vc,max, Jmax and ls.

thumbnail Figure 1

Mesophyll conductance to CO2 (gmes), per treatment (control, C; winter resprouts, RW; summer resprouts, RS) and season (winter, summer). In summer, the time of day was included (morning, midday, evening). Values are mean ±SE of 10 replicates. Significant differences (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and, in summer, time of day (α, β, γ).

Table IV

CO2 concentration in the chloroplast (CC) and midday mesophyll conductance and stomatal conductance ratio (gmes / gs) at Ca = 350 μmol mol−1 from A/Ci curves for different treatments (control, C; winter resprouts, RW; summer resprouts, RS) and season (winter, summer). In summer, the time of day was also considered for CC values. Each value represents the mean ±SE of 10 replicates. Significant differences (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and time of the day (α,β,γ)).

3.2. Mesophyll conductance

In winter, no difference between treatments was observed in midday gmes. In summer, R showed higher daily values than C (Fig. 1). Morning values were 36.1% higher than at midday and in the evening. gmes values at midday declined by 97% in controls from winter to summer; whereas in R values declined by 76%. In both seasons, no significant difference in CC values between treatments was found (Tab. IV). At midday, the gmes / gs ratio was higher in winter than in summer in both kinds of plant, whereas no difference was found between treatments in the two seasons (Tab. IV).

thumbnail Figure 2

Leaf mass per area (LMA), mean area of a leaf (LA), leaf density (D) and leaf thickness (T) per treatment (control, C; winter resprouts, RW; summer resprouts, RS) in winter and summer. Values are mean ±SE of 30 replicates. Significant differences (p ≤ 0.05) are indicated by different letters (treatment (a, b, c); season (A, B)).

3.3. Leaf growth parameters

LMA showed no seasonal change. In winter, LMA, D and T were higher in C (Figs. 2a, 2c, 2d), whereas in summer, R showed lower LMA and D but higher T. No seasonal difference in density and thickness was found in C. Mean leaf area (LA) was higher in R and decreased from winter to summer (Fig. 2b). LMA and D were negatively related to gmes; whereas for T the relationship was positive (Figs. 3a–3c).

thumbnail Figure 3

Mesophyll conductance to CO2 (gmes) vs. leaf mass per area (LMA) (a), leaf density (D) (b) and leaf thickness (T) (c) per season (winter, black; summer, white). Symbols represent single measurements of: control (C; •, ◦); winter resprouts, (RW; ▲, ∆); summer resprouts, (RS; ∇). Asterisks indicate statistically significant correlations (* p ≤ 0.05).

3.4. Leaf carbon isotope composition

Isotope discrimination against 13C (Δ13C), calculated from δ13C data, was higher in R than in C for both seasons (Tab. V). Results for water-soluble extracts and dry matter showed the same trends. Δ13C showed a negative relationship with LMA and a positive relationship with gmes for both seasons (Figs. 4a, 4b). Δ13C showed a negative relationship with WUEi in the winter, whereas in the summer the relationship became positive (Fig. 4c).

Table V

Isotope discrimination against 13C (Δ13C), calculated from isotope composition data (δ13C). Data are shown according to treatments (control, C; winter resprouts, RW; summer resprouts, RS), material analysed (dry matter, DM; water-soluble extract, WSE) and season (winter and summer). Values are mean ±SE of 6 replicates. Significant differences across rows or columns (p ≤ 0.05) are indicated by different letters: treatment (a, b, c), season (A, B) and material analysed (α,β).

thumbnail Figure 4

Isotope discrimination against13C (Δ13C) of water-soluble extracts vs. leaf mass per area (LMA) (a), gmes (b) and instantaneous water use efficiency (WUEi = A / gs) (c) per season (winter, black; summer, white). Symbols represent single measurements of: control (C; •, ◦); winter resprouts, (RW; ▲, ∆); summer resprouts, (RS; ∇). Asterisks indicate statistically significant correlations (* p ≤ 0.05).

4. DISCUSSION

In summer, higher temperatures, irradiance and VPD and lower precipitation than in winter lead to increased drought in Mediterranean forests. In fact, gs, used as an integrative indicator for the degree of water stress (Galmés et al., 2007), showed resprout values corresponding to moderate water stress (gs = 100 − 150 mmol m−2 s−1), whereas water stress was severe for undisturbed plants (gs below 50 mmol m−2 s−1). Drought affected numerous measured parameters, declining by 20% in R and by 50% in C: A, Amax, diffusive conductance (gs and gmes), Vc,max, Jmax and ΦPSII. Higher values for R in the summer can be explained by the greater nutrient and water availability for small resprouting shoots than for controls as reflected both in higher gs and RWC. Moreover, the larger photosynthetic sink for electrons in R accounts for the lower thermal energy dissipation (estimated by the chlorophyll fluorescence parameter, NPQ) observed in summer, as reported elsewhere (Fleck et al., 1998). In contrast, environmental conditions in the winter did not induce differences in resprouts from undisturbed plants.

Resprout gmes was markedly higher (3.75-fold) than in C during summer drought. A decline in gmes with changes in plant water availability has been observed for other species (Roupsard et al., 1996). The obtained gmes values were lower than those reported for Q. ilex well-watered plants (Loreto et al., 1992). The absolute gmes values obtained in our study may be under-estimated as some parameters used in the calculations were not measured but assumed from the literature (leaf absorptance, light partition between photosystems I and II) or substituted by approximations (use of dark respiration instead of light respiration). However, our results are in the range obtained by Niinemets et al. (2005) for the same species in a forest study.

The 6-fold decrease in the ratio gmes / gs from winter to summer in controls and resprouts suggests a stronger photosynthesis limitation by gmes in Q. ilex than in previously published for other species (Niinemets et al., 2005). During water stress, Ci may be overestimated because of patchy stomatal closure, and consequently gmes would be underestimated. However, patchiness was not detected in this study (Materials and Methods, Leaf gas exchange).

The leaf structure of resprouts differed from controls and reflected their higher water availability: in fact, R showed higher mean leaf area and lower LMA, indicative of reduced water stress (Peña-Rojas et al., 2005).These structural characteristics may be primarily responsible for changes in gmes: the inverse relationship between gmes and LMA values (Fig. 3a) has been also reported (Niinemets et al., 2006). No seasonal change in LMA was observed in either kind of plants. However, in resprouts, a decrease in density and an increase in thickness were observed from winter to summer. These two components of LMA are not necessarily interdependent, and may be controlled by different environmental variables. However, high T is commonly associated with lower D (Mediavilla et al., 2001). In accordance with our results (Figs. 3b, 3c), gmes reduction has been related to increased D in peach (Syvertsen et al., 1995), and decreased T in spinach leaves grown under salt conditions (Delfine et al., 1998).

Lower D and higher T in resprouts may also account for the observed increased photosynthesis because they correlate with air space fraction in the mesophyll (Niinemets, 1999) resulting in higher gmes. Moreover, T is linearly related to the surface area of cells exposed to intercellular air spaces per unit leaf area (Hanba et al., 2002). As chloroplasts are usually distributed near the cell surface, the T increase in R accounts for higher photosynthetic protein accumulation per unit leaf area.

However, morphology is not the only factor determining gmes since the strong reduction in gmes from winter to summer in controls was not paralleled by a change in LMA, T or D; and daily changes in gmes in the summer cannot be attributed to changes in leaf morphology, either. gmes responds not only in the long term to environmental stress, but also changes within seconds to minutes even faster than gs does (Flexas et al., 2008). Short-term changes in gmes have been attributed to carbonic anhydrase (Gillon and Yakir, 2000) and chloroplast aquaporin regulation (Terashima and Ono, 2002; Flexas et al., 2007). Thus, our results are consistent with the idea that gmes is grossly determined by leaf structure, but is also the result of physiological control. In our study, the daily variations in gmes were of the same magnitude as the seasonal variations, indicating that gmes regulation might be as important as the constraints imposed by morphology.

gmes variations in both kinds of plant paralleled changes in A and Amax, which may indicate a reduction of photosynthesis in response to sustained low chloroplast CO2 levels (Flexas et al., 2006). However, a limitation of photosynthesis not directly related to CO2 diffusion is suggested by the analysis of A / Ci curves. The decreases in A and Amax in all treatments from winter to summer were paralleled by those of Vc,max and Jmax, indicating a non-stomatal limitation of photosynthesis. In R, this limitation was lower; they showed around 50% higher Vc,max and Jmax. These results are compatible with a down-regulation of CO2 assimilation to adjust mesophyll capacity to the decreased CO2 supply due to gs and gmes effects (Flexas et al., 2006). This adjustment of the mesophyll capacity would result in maintenance of Cc as observed (Tab. IV), in the same way that Ci (Tab. II) tends to remain constant, as reported by Wong et al. (1979).

Morphological and physiological changes during drought can be reflected in Δ13C values: we observed a negative relationship between Δ13C and LMA values (Fig. 4a), as reported (Fleck et al., 1996). This trend may be a consequence of a gs decline, but can also be due to a gmes decline. A significant contribution of internal resistances to foliar Δ13C has been proposed for other species owing to its effect on CO2 partial pressure at the carboxylation site (Vitousek et al., 1990) and is reflected in Figure 4b. The expected, negative relationship between WUEi and Δ13C observed in winter values reflects a similar contribution of gs and gmes to A and Δ13C, resulting in WUEi increasing as gs decreases (A decreases less than gs because of the sustained consumption by RuBisCO) and in Δ13C decreasing as gs decreases (because of Ci decline).

The positive relationship observed in summer, (Fig. 4c) can be explained by a dominant role of gmes, mainly in C: here, A declines more than gs, especially in some plants, because of the strong reduction in gmes, resulting in a decrease in WUEi, and not in its increase, as expected as gs decreases (Tab. II). Meanwhile, the sum of reduced gs and gmes, caused the expected decrease in Δ13C, resulting in the observed positive relationship with WUEi. Interestingly, gmes reduction has been proposed as an explanation for the inability of typical gas exchange models to predict WUE in Mediterranean ecosystems (Reichstein et al., 2002). In fact, Warren and Adams (2006) proposed, from a theoretical point of view, that gmes may affect the relationship between Δ13C and WUE. Such a disagreement was not found by Roussel et al. (2009) in Quercus robur, but Flexas et al. (2008) already found a discrepancy between WUE and δ13C that could be attributed to changes in gmes, between transgenic tobacco plants, but ours is the first report of a clear mismatch between Δ13C and WUE in forest growing plants that can be attributed to gmes. The original Δ13C model (Farquhar et al., 1982) already included a term for gmes that is often ignored in typical models, but should be included for prediction of the absolute value of leaf Δ13C.

We conclude that gmes exerts a dominant role in photosynthesis limitation in Q. ilex. A regulation of gmes exists beyond the morphological constraints, and both factors may well be of a similar magnitude. The greater capacity of resprouts to withstand drought that implied lower photosynthetic limitants (both diffusive and non-stomatal) will permit their growth and recovery after increased fire episodes associated with the climate change.

Acknowledgments

This work was supported by funds from the Generalitat de Catalunya (2001SGR00094). We thank L. Cabañeros, J. Vilamú and the Can Coll team (Parc Natural de Collserola) for site facilities; the Servei de Camps Experimentals, UB, for technical assistance; and R. Rycroft and assistants (Servei d’Assesorament Lingüístic, UB) for correcting the English manuscript. K. Peña-Rojas was the recipient of a doctorate grant from AECI and from the Faculty of Forestry Engineering, University of Chile.

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All Tables

Table I

Climatological data recorded at the forest site during the gas-exchange measurements of the different treatments (control, C; winter resprouts, RW; summer resprouts, RS); data are the mean ±SE of all measurements.

Table II

Midday values of net photosynthesis (A), stomatal conductance (gs), intercellular CO2 concentration (Ci), instantaneous water use efficiency (WUEi = A / gs), relative water content (RWC), PSII efficiency (ΦPSII), non-photochemical quenching of fluorescence (NPQ) and potential quantum yield of PSII at midday (Fv / Fm). Data are presented according to treatment (control, C; winter resprouts, RW; summer resprouts, RS), leaf orientation (north, south) and season (winter, summer). Values are mean ±SE of nine replicates. Significant differences across rows or columns (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and leaf orientation (α,β)).

Table III

Net CO2 assimilation at saturating Ci and light (Amax), maximum carboxylation velocity of Rubisco (Vc,max), maximum potential rate of electron transport contributing to RuBP regeneration (Jmax) and stomatal limitation (ls) from A/Ci curves for the different treatments (control, C; winter resprouts, RW; summer resprouts, RS) and season (winter, summer). In summer, the time of day was also considered. Each value represents the mean ±SE of ten replicates. Significant differences across rows or columns (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and time of the day (α,β,γ)).

Table IV

CO2 concentration in the chloroplast (CC) and midday mesophyll conductance and stomatal conductance ratio (gmes / gs) at Ca = 350 μmol mol−1 from A/Ci curves for different treatments (control, C; winter resprouts, RW; summer resprouts, RS) and season (winter, summer). In summer, the time of day was also considered for CC values. Each value represents the mean ±SE of 10 replicates. Significant differences (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and time of the day (α,β,γ)).

Table V

Isotope discrimination against 13C (Δ13C), calculated from isotope composition data (δ13C). Data are shown according to treatments (control, C; winter resprouts, RW; summer resprouts, RS), material analysed (dry matter, DM; water-soluble extract, WSE) and season (winter and summer). Values are mean ±SE of 6 replicates. Significant differences across rows or columns (p ≤ 0.05) are indicated by different letters: treatment (a, b, c), season (A, B) and material analysed (α,β).

All Figures

thumbnail Figure 1

Mesophyll conductance to CO2 (gmes), per treatment (control, C; winter resprouts, RW; summer resprouts, RS) and season (winter, summer). In summer, the time of day was included (morning, midday, evening). Values are mean ±SE of 10 replicates. Significant differences (p ≤ 0.05) are indicated by different letters (treatment (a, b, c), season (A, B) and, in summer, time of day (α, β, γ).

In the text
thumbnail Figure 2

Leaf mass per area (LMA), mean area of a leaf (LA), leaf density (D) and leaf thickness (T) per treatment (control, C; winter resprouts, RW; summer resprouts, RS) in winter and summer. Values are mean ±SE of 30 replicates. Significant differences (p ≤ 0.05) are indicated by different letters (treatment (a, b, c); season (A, B)).

In the text
thumbnail Figure 3

Mesophyll conductance to CO2 (gmes) vs. leaf mass per area (LMA) (a), leaf density (D) (b) and leaf thickness (T) (c) per season (winter, black; summer, white). Symbols represent single measurements of: control (C; •, ◦); winter resprouts, (RW; ▲, ∆); summer resprouts, (RS; ∇). Asterisks indicate statistically significant correlations (* p ≤ 0.05).

In the text
thumbnail Figure 4

Isotope discrimination against13C (Δ13C) of water-soluble extracts vs. leaf mass per area (LMA) (a), gmes (b) and instantaneous water use efficiency (WUEi = A / gs) (c) per season (winter, black; summer, white). Symbols represent single measurements of: control (C; •, ◦); winter resprouts, (RW; ▲, ∆); summer resprouts, (RS; ∇). Asterisks indicate statistically significant correlations (* p ≤ 0.05).

In the text