Trees (2010) 24:1029–1043 DOI 10.1007/s00468-010-0472-3
ORIGINAL PAPER
Microclimatic conditions determined by stem density influence leaf anatomy and leaf physiology of beech (Fagus sylvatica L.) growing within stands that naturally regenerate from clear-cutting Iva´n Closa • Juan Jose´ Irigoyen • Nieves Goicoechea
Received: 17 March 2010 / Revised: 21 June 2010 / Accepted: 20 July 2010 / Published online: 10 August 2010 Ó Springer-Verlag 2010
Abstract Beech forests naturally regenerating from clear-cutting can exhibit different microclimates depending on size of saplings and stem density. When beech trees are young and stem density is low, the level of radiation inside the ecosystem reaching the soil surface is high; consequently, air and soil temperatures rise and the soil water content may decrease. These microclimatic parameters presumably will affect the anatomy, photosynthesis, and carbon metabolism of beech leaves. We studied the morphology and physiology of sun and shade leaves of beech trees differing in age and growing within clear-cut areas with distinct microclimate. Results were compared with those of adult trees in an unmanaged forest. We selected a stand clear-cut in 2001 (14,000 trees ha-1), another clearcut in 1996 (44,000 trees ha-1) and an unmanaged forest (1,000 trees ha-1). Photosynthetic photon flux density (PPFD) incident on sun leaves, air temperature, soil moisture, and soil temperature within the forests affected water status and carbohydrate storage in all trees. As trees became older, PPFD also influenced pigment composition and Rubisco activity in sun leaves. On the other hand, shade leaves from the oldest trees were the most sensitive to PPFD, air temperature, and soil moisture and temperature inside the forest. Contrariwise, microclimatic parameters slightly affected the physiology of shade leaves of the beech in the stand with the highest light attenuation. Air and soil temperatures were the parameters that most Communicated by M. Buckeridge. I. Closa J. J. Irigoyen N. Goicoechea (&) Departamento de Biologı´a Vegetal, seccio´n Biologı´a Vegetal (Unidad Asociada al CSIC, EEAD, Zaragoza e ICVV, Logron˜o), Facultades de Ciencias y Farmacia, Universidad de Navarra, c/Irunlarrea 1, 31008 Pamplona, Spain e-mail:
[email protected]
affected the photosynthetic pigments and carbohydrate storage in shade leaves of the youngest trees. Keywords Beech Leaf anatomy Leaf physiology Microclimate Natural regeneration Rubisco
Introduction European beech (Fagus sylvatica L.) is a prevalent tree species throughout western and central Europe. In the Mediterranean basin, beech is confined to mountainous regions where rainfall is high (Garcı´a-Plazaola and Becerril 2000). In Spain, the most extensive beech forests, around 140,000 ha, are located in Navarra, a region in the northwest part of the Pyrenees. They represent 37% of the beech forests in the whole country. The distribution area of F. sylvatica in the province of Navarra ranges from the western end of the Pyrenees and the French border in the north to the lower areas around the Ebro river basin under Mediterranean influence to the south (Amores et al. 2006). However, many beech forests in Navarra have been deeply disturbed as a consequence of some management practices, such as pastures establishment and clear-cutting. When logs are removed by using heavy machinery, soil compaction and loss of seedlings, seeds, and nutrients may further damage plant habitats and reduce the capacity for forest regeneration (Van der Hout 2000). Regeneration of several beech forests subjected to clear-cutting in Navarra has been left to occur from the seed bank and from seeds provided by the surrounding beech trees. Natural regeneration is the process by which trees and woodlands are established from seeds produced and germinated in situ. One of the advantages of natural regeneration when compared with planting is that it can conserve local genotypes
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and also create more structural diversity within the site (Harmer and Gill 2000). The use of natural regeneration has been increasing for the last three decades in part due to increased awareness among foresters of the environmental and conservation benefits that natural regeneration can bring (see Harmer et al. 1997). However, several criteria should be considered when applying natural regeneration (Evans 1988; Harmer et al. 1997): the quality and suitability of the parent crop; supply of viable seed; the presence of advance regeneration seedlings, which are present before harvesting of the mature trees; suitability of the forest floor for germination; survival and establishment of young seedlings; competition from weeds and browsing damage. Several studies have focused on natural regeneration of deciduous (i.e., Harmer et al. 1997, 2005; Harmer and Morgan 2007), coniferous (see Kerr 2000 for review) or evergreen broad-leaved (i.e., Wu et al. 2008) forests. In deciduous trees, and in particular in beech, under natural regeneration the sapling stage is usually followed by a transition thicket stage prior to development of a young high-growing forest, that usually is initiated in the forestry practice by selective clearing measures. However, there can be several factors—i.e., poor nut production by surrounding trees, adverse climatic conditions or browsing damage—that may decrease survival or growth rate of young beech trees within clear-cut areas, which occurred in one of the beech stands, in the present study, that was clearcut in 2001. As a consequence, the common pattern of natural regeneration would be altered, microclimatic conditions would be affected by the unexpected low number of beech trees and, vice versa, microclimate within the disturbed area would influence the physiology and development of the young beech trees. Among the parameters presumably strongly affected by microclimatic conditions would be those related to anatomy, photosynthesis, and carbon metabolism in sun and shade leaves. Therefore, the main objective of our work was to deepen the knowledge of some anatomical, physiological, and biochemical parameters of both sun and shade leaves of beech trees that had successfully established within clearcut areas exhibiting very different tree density and, consequently, strong differences in microclimatic conditions. These anatomical, physiological and biochemical parameters were compared with those of mature trees within an unmanaged forest.
Materials and methods Field site Beech forests were located in Oderitz (northwest Navarra, Spain; 42°580 , 1°520 ; al. 750 m). The climate in the area is
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classified as temperate, humid coastal-type with an average annual temperature that ranges between 11 and 14.5°C and an annual precipitation between 1,100 and 2,500 mm. Fagus sylvatica L. tends to dominate in the mountain slopes above 600 m a.s.l., whereas the low lying areas are populated by other tree species such as Quercus robur L. (oak). The lithology in the selected area is predominantly calcareous and the beech stands chosen for this study belonged to the series Carici sylvaticae-Fageto sylvaticae S. according to Loidi and Ba´scones (2006). However, the abundant and frequent rain in this area favored the acidic pH of the soil (Table 1). Three beech stands were selected for this study: (i) a forest that was unmanaged for at least one century, (ii) a beech stand that was clear-cut in 1996, and (iii) a beech stand subjected to clear-cutting in 2001. The main characteristics of each beech stand, the average height and diameter at breast height (DBH) of dominant and codominant trees and soil characteristics within each stand are given in Table 1. Fagus sylvatica was the only woody species present in the forest that was clear-cut in 2001 and was also the predominant tree in the other forests under investigation. There was also a specimen of Ilex aquifolium L. within the stand forest subjected to clear-cutting in 1996 and Corylus avellana L. was present within the unmanaged forest. The three forests were surrounded by F. sylvatica. While the stem density was extremely high within the stand clear-cut in 1996 (44,000 trees ha-1), the lowest stem density corresponded to the unmanaged forest (1,000 trees ha-1), the stand with the biggest and the oldest beech trees. The stem density in the stand clear-cut in 2001 was 14,000 trees ha-1. Soil collected from the stand disturbed in 2001 had lower pH, organic matter, available phosphorus (P), total organic nitrogen (N) and cation exchange capacity (CEC) and also had a coarser texture than soils from the stand clear-cut in 1996 and the unmanaged beech forest. Maximum and minimum temperatures as well as monthly total precipitation from August 2007 to October 2008 were measured at the station of Alli, 8 km far away from the study area (Fig. 1). Temperatures reached or exceeded 30°C in August 2007 and June, July and August 2008. Freezing temperatures were recorded in November and December 2007 as well as in January, February, and April 2008. The maximum temperature (36°C) was achieved in July 2008 and the minimum temperature (-6°C) was reached in November 2007. The highest accumulation of rainfall was recorded in March 2008 (400 mm). In contrast, February, July, August and September 2008 were the driest periods, with less than 50 mm accumulated in each month. Clear-cutting consisted of the complete removal of all trees [8 cm in DBH in a single harvest using a chain saw.
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Table 1 Basic characteristics of the studied beech stands
Clear-cut in 2001
Clear-cut in 1996
Unmanaged
Former stand
Fagus sylvatica
Fagus sylvatica
Fagus sylvatica
Surrounding trees
Fagus sylvatica
Fagus sylvatica
Fagus sylvatica
Tree age
Around 8 years
Around 13 years
[100 years
Max. tree height (m)
2.5
5.10
31.2
DBH (cm)
8.76
23.12
94.74
Stem density (no. ha-1)
14,000
44,000
1,000
Vegetation
Soil pH (H2O)
5.3
5.1
5.3
P (mg kg-1)
8.4
14.6
10.8
N (g-%)
0.30
0.63
0.53
CEC (cmol (?) kg-1)
28.6
46.8
41.7
Organic matter (g-%)
7.9
15.9
12.0
C/N
11
15
14
28.4
10.1
13.0
Silt (%)
42.1
62.7
51.5
Clay (%)
29.5
27.3
35.6
Texture Sand (%)
Physicochemical parameters of soils
40 35
500
25 400
20 15
300
10 200
5 0
Precipitation (mm)
Temperature (°C)
30
Precipitation Maximum temperature Minimum temperature
100
-5 -10
A S O N D J F M A M J J A S O 2007
0
2008
Fig. 1 Maximum (filled square) and minimum (open square) air temperatures (°C) and rainfall (mm) monthly accumulated from August 2007 to October 2008
All trees were felled at the base, approximately 0.20 m above the soil surface. The felled trees were subsequently transported by heavy machinery. Soil scarification was not applied after trees were cut, beech trees were not planted in the clear-cut areas, and sprouting from cut trees was not observed in any case. Natural regeneration occurred from the remaining seeds in the disturbed areas and from the seeds provided by the surrounding beech trees. Nine dominant or co-dominant beech trees were selected within each stand for further physiological and biochemical determinations.
The soil type in the area is a Typic Eutrudept (USDA 1999). Within every beech stand, nine soil samples of 500 ml to a depth of 10 cm starting at the top of the A horizon (one soil sample per plot) were collected and homogenized to obtain a uniform sample of the physicochemical characteristics of each site as a whole. ‘O’ horizons were not present. In order to minimize effects of stemflow water on physicochemical characteristics (Matschonat and Falkengren-Grerup 2000), soil samples were taken 1–2 m far away beech trees. Physicochemical analyses of the soils from the three studied beech stands were performed as described by Closa and Goicoechea (2010). Soil water content, soil temperature, photosynthetic photon flux density (PPFD), leaf area index (LAI) and air temperature Soil moisture at a depth of 10 cm was gravimetrically estimated (Bethlenfalvay et al. 1990). Soil temperature was measured at a depth of 10 cm in the field with a Testo 110 thermometer (Testo AG, Lenzkirch, Germany). Photosynthetic photon flux density (PPFD) (lmol m-2 s-1) was measured inside and outside each beech stand at 1.5 m from the soil surface by using a radiometer (Li-Cor, LI188B model, Lincoln, NE, USA). Sun leaves were exposed to a maximum PPFD of 1,300 lmol m-2 s-1. Shade leaves of 8-, 13-year-old and mature trees received up to 300, 20 and 100 lmol m-2 s-1, respectively, at the inner tree
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crown. Leaf area index (LAI) was measured with a ceptometer (AccuPAR LP-80 model, Decagon Devices, Inc., Pullman, WA, USA). The air temperature outside and inside each beech stand was also measured at a distance 1.5 m above ground level using a Testo 110 thermometer (Testo AG, Lenzkirch, Germany). Leaf water status and specific leaf area (SLA) Sun and shade leaves of beech trees were handy sampled in autumn (25 October 2007), spring (28 May 2008), and summer (3 September 2008) from the nine trees selected within each stand. Leaves were immediately transferred to the laboratory in individual sealable plastic bags on ice. Once there, leaves free of apparent damage were randomly selected to calculate their fresh weight (FW). Turgid weight (TW) was calculated after fully hydrating fresh leaves in darkness at 4°C for 24 h. Leaf dry weight (DW) was determined after drying at 80°C for 2 days. Leaf water content was calculated as 100 9 (FW - DW)/DW. Relative water content (RWC) of leaves was estimated by Weatherley’s method (1950) and calculated as RWC = 100 9 (FW - DW)/(TW - DW). Individual leaf area was recorded by using an automatic leaf area meter (Li-Cor, LI-3000 model, Lincoln, NE, USA). Specific leaf area (SLA) was calculated as the ratio of individual leaf area to leaf dry matter (leaf DW).
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pigments. The absorbance of extracts was measured at 470, 649, 665, and 750 nm using a U-2001 spectrophotometer (Hitachi Instruments, Inc., USA). Estimation of Chl a, Chl b and total Cars in the same extract solution was performed by using the extinction coefficients and equations redetermined by Lichtenthaler (1987). From the pigments levels the weight ratios of pigments, Chl a/b and Chls/Cars (a ? b)/(x ? c) were determined. Soluble proteins, total soluble sugars (TSS) and starch in leaves Leaves were collected as described previously. Total soluble proteins, TSS and starch concentration were quantified in potassium phosphate buffer (KPB) (50 mM, pH 7.5) extracts of fresh leaves (250 mg). These extracts were filtered through four cheese cloth layers and centrifuged at 28,710 g for 15 min at 4°C. The supernatant was collected and stored at 4°C for protein and TSS determinations. The pellet was used for starch determination (Jarvis and Walker 1993). Leaf soluble protein was measured by the protein dye-binding method of Bradford (1976) using bovine serum albumin (BSA) as standard. Total soluble sugars (TSS) were analyzed with the anthrone reagent in a U-2001 spectrophotometer (Hitachi Instruments, Inc., USA) (Yemm and Willis 1954). Rubisco activity assay (EC 4.1.1.39)
Anatomical characteristics of leaves Dental resin impressions were used to study the anatomical characteristics of abaxial leaf surfaces. Fresh resin was applied at the point of maximum leaf width near the central vein. The dental resin mold was filled with nail polish to create a cast that was examined under a light microscope (Nikon Eclipse E200, Nikon Instruments Inc., Melville, NY, USA) (409) according to Geisler et al. (2000). From each stand, 45 sun leaves and 45 shade leaves and five microscopic fields of each epidermal surface impression were randomly examined. Stomata index was calculated as described by Cutter (1969): stomata index = NS/ (EC ? NS), where NS is the number of stomata and EC is the number of epidermal cells per unit leaf area. Results on stomata index were expressed as percentages.
Leaves were collected as described previously. Rubisco (EC 4.1.1.39) extractable activity was measured by grinding and filtering 250 mg of leaf fresh weight. The leaf tissue was powdered in liquid nitrogen and homogenized in a cold mortar with an extraction buffer containing 100 mM Bicine-NaOH (pH 7.8), 10 mM MgCl2, 10 mM 2-bmercaptoethanol, 2% polyvinylpolypyrrolidone (PVPP) (w/v), 1% BSA (w/v) and 1% Triton X-100 (v/v). The extract was clarified by centrifugation at 26,850 g for 10 min at 4°C and then stored at -80°C after adding 20% (v/v) glycerol in order to protect Rubisco enzyme. Rubisco activity was determined in 100 lL aliquots by measuring the oxidation of nicotinamide adenine dinucleotide (NADH) at 340 nm (U-2001 spectrophotometer, Hitachi Instruments, Inc., USA), as described by Sharkey et al. (1991).
Pigment analysis Transpiration decline curves (TDC) Leaves were collected as described previously. The photosynthetic plant pigments, total chlorophylls (Chl a ? b) (Chls) and total carotenoids (Cars) (x ? c, xanthophylls ? carotenes) were extracted according to Se´stak et al. (1971). Samples (3 mg FW) were immersed in 5 mL of 96% ethanol at 80°C during 10 min to extract the
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Branches from sun and shade conditions were sampled in autumn (25 October 2007), spring (28 May 2008) and summer (3 September 2008) from the nine trees selected within each stand and immediately transferred to the laboratory in individual plastic bags on ice. Once in the
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laboratory, leaves from the middle part of the branches (five leaves per branch) were excised in order to obtain leaf transpiration curves following the methodology described by Darwish and Fahmy (1997). As soon as possible after excision, the leaf petiole was placed in water with its leaflets in air at 100% relative humidity (RH). The leaves were allowed to rehydrate overnight in the dark at 4°C. On the following day, the water-saturated leaves were weighed to calculate the TW and then they were exposed to diffuse natural light for 90 min at 20°C and 100% RH to induce stomata to open. In order to know if stomata had opened after this period the other sun and shade leaves (n = 7–9) from each beech stand were also subjected to rehydration overnight in the dark at 4°C and exposed to diffuse natural light for 90 min at 20°C and 100% RH on the following day. Afterwards, these leaves were used to obtain dental resin impressions from the abaxial leaf epidermis for measuring the length and width of the stomatal pores under a light microscope (Nikon Eclipse E200, Nikon Instruments Inc., Melville, NY, USA) (1,0009). The length of the stomatal pores ranged from 15.2 lm in sun leaves of the beech trees in the stand clear-cut in 2001 to 26.5 lm in sun and shade leaves of the beech trees in the stand clearcut in 1996. These values are similar to those measured by Cˇanˇova´ et al. (2008) in several European beech cultivars. The width of the stomatal pores in sun and shade leaves achieved values that ranged from 68 to 100% of those corresponding to length, which indicated that stomata were open before calculating the transpiration decline curves. The water-saturated leaves selected for the transpiration decline curves were superficially dried with soft tissue paper and exposed to light (photosynthetic active radiation, PAR = 180 lm m-2 s-1) in a controlled environment chamber (Conviron PGV 36, Winnipeg, CA, USA) at 25 ± 0.5°C and 40–50% RH. The rate of water loss was determined for each leaf by weighing it on an electronic balance (Mettler Toledo AB104, Greifensee, Switzerland) at 10 min intervals for the first hour, 15 min intervals for the second hour, 20 min intervals for the third hour, 30 min intervals for the fourth hour and, for leaves collected both in autumn and summer, 1 h interval for the following 3 h to obtain fresh mass at each moment. Afterwards, leaf DW was determined by drying at 80°C for 48 h. Both leaf RWC and rate of water loss were inferred from the data. RWC versus rate of water loss curves were plotted and asymptotic exponential functions were fitted to data. However, in order to calculate the value of RWC at which stomatal closure occurred, it was necessary to apply linear regression analysis to the points. Only for leaves collected in spring, data clearly fitted the single inflection model with two markedly different stages described by Quisenberry et al. (1982). We calculated the slope at each stage through linear regression analysis including those points that
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graphically showed to belong to each stage. The slope of the first stage can be considered to represent the rate of total mostly stomatal transpiration, and the second slope the water loss through the cuticle and the imperfectly closed stomata, whereas the point of inflection allows estimating the value of RWC at which the mean stomata closure takes place. For leaves collected in autumn and summer, curves did not show inflection (Quisenberry et al. 1982). Statistical analysis All analyses were performed using the statistical package SPSS (version 12.0) (SPSS, Chicago, IL) for Windows xp (Microsoft, Seattle, WA). Every beech stand was considered as an experimental unit. Data were tested for homogeneity of variances using Mauchly’s Test of Sphericity. Where data sets failed Mauchly’s Test of Sphericity (data on PPDF), differences of parameters across the year were tested using the F value generated by Pillai’s trace. When sphericity could be assumed (all the measured parameters except for PPFD), the Huyn–Feldt statistic was used for within-subject tests, being ‘‘season of the year’’ the withinsubject factor with four levels. Bonferroni was applied for post-hoc testing and confidence interval estimation. Pearson analyses were made to test the possible correlations between the different microclimatic parameters within each beech stand—PPFD, air temperature, soil water content, and soil temperature—and to evaluate the effect of those microclimatic parameters on the physiological and biochemical parameters of both sun and shade leaves. Significant levels were always set at 5.0% level of significance (Figs. 2–8, Table 2).
Results Soil moisture reached a maximum value in spring within the stand clear-cut in 2001 (0.41 g H2O g-1 dried soil) and remained comparable across the seasons within the stand clear-cut in 1996 (achieving mean values of around 0.66 g H2O g-1 dried soil) and showed a marked seasonal course within the unmanaged forest (Table 2). Within the unmanaged forest, soil water content reached a maximum value in spring (0.57 g H2O g-1 dried soil), a minimum value in summer (0.26 g H2O g-1 dried soil), and an intermediate level in autumn (0.42 g H2O g-1 dried soil). Photosynthetic photon flux density (PPFD), as expected, was always higher in sun than in shade leaves regardless of beech stand and season of the year (Table 2). In autumn, the difference of PPFD between sun and shade leaves was similar within the three studied stands. However, in spring, this difference was especially small in the youngest trees
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1034 600 500
a1
Sun Shade
a2 a
400
200
a
a
300
b
a
d
abc bcd
ab cd
10 5
SLA (cm2 g-1 DM)
0 500
b
400
c
300
d
d
a
b1
a
b b
b
b2 b
b
0 30 25 20 15
d
10
100
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0
c1
500
c2
a b c
300
d 200
d
a
ab
400
ab b
ab
ab
0 30 25 20 15
d
10
100
5
0 autumn 2007
spring 2008
from the stand clear-cut in 2001. In spring, the strongest attenuation of light occurred within the stand disturbed in 1996. In summer, the greatest difference in PPFD between sun and shade leaves corresponded to the oldest trees within the unmanaged forest. The lowest leaf area index (LAI) was measured within the stand clear-cut in 2001 through the year (Table 2). LAI reached a maximum value in spring and a minimum value in autumn within the three studied stands and, except for the stand clear-cut in 2001, LAI significantly declined from spring to summer. The air temperatures within the three studied beech stands followed a similar pattern across the year, achieving minimum values in autumn, intermediate values in spring and the highest values in summer (Table 2). Specific leaf area (SLA) was comparable among sun leaves of different trees collected at different seasons of the year and it reached values varying from 170 to 200 cm2 g-1 DM (Fig. 2a1, b1, c1). On the other hand, SLA was always higher in shade than in sun leaves regardless of tree age and leaf phenological stage. However, while the difference in SLA between sun and shade leaves was similar through the life cycle in the youngest trees (Fig. 2a1), that difference achieved maximum values in spring for leaves of both the 13-year-old and the oldest trees, and then it progressively decreased from spring to
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20 15
100
200
30 25
b
b
35
Individual leaf area (cm2)
Fig. 2 Specific leaf area (SLA) (cm2 g-1 DM) (a1, b1, c1) and individual leaf area (cm2) (a2, b2, c2) of sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clear-cut in 2001 (a1, a2), the stand clearcut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05). DM dry matter
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summer 2008
autumn 2007
spring 2008
summer 2008
0
summer (Fig. 2b1, c1). Individual leaf area of sun and shade leaves in the youngest trees was comparable in spring; however, leaf area of shade leaves was lower than that of sun leaves in summer and autumn (Fig. 2a2). In contrast, leaf area of shade leaves in the 13-year-old trees was higher than that of sun leaves in spring and it was comparable between both types of leaves in other seasons of the year (Fig. 2b2). Only in the oldest trees individual leaf area was similar between sun and shade leaves through the life cycle (Fig. 2c2). Stomata index reached values of around 30% in both sun and shade leaves of the youngest trees that grew within the stand clear-cut in 2001 (Fig. 3a1). In the 13-year-old trees, belonging to the stand disturbed in 1996, the stomata index of sun leaves also achieved values near 30%, and stomata index in shade leaves varied from 15% in senescent leaves (autumn) to 20% in young leaves (spring) and mature leaves (summer) (Fig. 3b1). The highest stomata index was measured in sun leaves of the oldest trees and reached values near 35% (Fig. 3c1). However, stomata index in shade leaves of the oldest trees varied from 15 to 18%. Consequently, the greatest difference in stomata index between sun and shade leaves corresponded to beech trees growing within the unmanaged forest (Fig. 3c1). Stomata density of sun leaves was higher than 200 stomata mm-2 regardless of
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40 35 30
Sun Shade a ab
a1 ab ab
ab
300
b
b
b
25
a2 350
a
a
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c
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200
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Stomata Index (%)
0
a
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a
ab bc
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b1
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b
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c
10
100 50
5 0 35
a
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25 20 15
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b
0 300 250 200
c d
d
150 100
10
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5 0
300 250
a
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0
Stomata density (stomata mm-2)
Fig. 3 Stomata index (%) (a1, b1, c1) and stomata density (no. stomata mm-2) (a2, b2, c2) of sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clear-cut in 2001 (a1, a2), the stand clear-cut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05)
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autumn 2007
spring 2008
tree age and phenological stage of leaves (Fig. 3a2, b2, c2). However, while stomata density in sun leaves from the 13year-old beech trees remained almost unchanged across the seasons of the year (Fig. 3b2), this parameter decreased from spring to summer in sun leaves of the youngest trees (Fig. 3a2) and achieved the highest value in autumn in sun leaves of the oldest trees (Fig. 3c2). In general terms, stomata density of shade leaves was always lower than that of sun leaves, independent of tree age and phenological stage of leaves (Fig. 3a2, b2, c2) and showed marked seasonal changes. In fact, the greatest values of stomata density in both the youngest (Fig. 3a2) and the oldest (Fig. 3c2) trees were observed in senescent leaves (autumn). In the 13-yearold trees, stomata density progressively decreased as leaves were senescing (Fig. 3b2). In spring, when new leaves were developing, the content of total chlorophylls (Chls) (a ? b) per unit of leaf area in sun and shade leaves clearly differed among beech trees of different age (Fig. 4). While the amount of Chl a ? b was significantly higher in shade than in sun leaves of the youngest trees (Fig. 4a1), both types of leaves had similar levels of Chl a ? b in the 13-year-old trees (Fig. 4b1) and, in the oldest trees, the highest content of Chl a ? b was found in sun leaves during spring (Fig. 4c1). However, in
summer 2008
0 autumn 2007
spring 2008
summer 2008
summer, and except for the youngest trees, shade leaves had greater content of total Chls than sun leaves. In autumn, sun and shade leaves had similar levels of total Chls regardless of tree age. The levels of total carotenoids (Cars) (x ? c) exhibited a similar pattern than that of total chlorophylls in spring (Fig. 4). The contents of Cars in shade leaves were higher (Fig. 4a2), similar (Fig. 4b2) or lower (Fig. 4c2) than those in sun leaves in the youngest, the 13-year-old and the oldest trees, respectively. In summer and autumn, the levels of Cars were lower in shade than in sun leaves independent of tree age. The ratio Chl a/b was always lower in shade (Chl a/b = 2–2.7) than in sun (Chl a/b = 2.8–3.2) leaves collected from the 8- and 13-year-old trees (Fig. 5a1, b1). On the other hand, that ratio was similar (Chl a/b = 2.4–2.6) in sun and shade of leaves of the oldest trees in both spring and autumn, coinciding with the youngest and the senescent stages of leaves, respectively (Fig. 5c1). The total Chls to total Cars ratio reached higher values in shade (Chls/Cars = 4.7–7.8) than in sun (Chls/Cars = 2.9–4.4) leaves of both 8- and 13-year-old trees (Fig. 5a2, b2). In contrast, the ratio Chls/Cars was similar in both types of leaves in the oldest trees in autumn and spring (Fig. 5c2). The maximum value of the Chls/Cars ratio was measured
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1036 450
a1
a
375
a2 a ab
300 225
b
bc bc
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80 60
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150 20 75 0
Chls (a+b) (mg m-2)
0
b1
375 300
ab ab
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a
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300 225
Cars (x + c)) (mg cm-2)
Fig. 4 Total chlorophylls (Chls) (a ? b) (mg m-2) (a1, b1, c1) and total carotenoids (Cars) (x ? c) (mg cm-2) (a2, b2, c2) in sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clear-cut in 2001 (a1, a2), the stand clear-cut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05). x xanthophylls, c carotenes
Trees (2010) 24:1029–1043
bc cd
c2 a
ab
60
bc
bc d
cd
d
d
40
150 20 75 0
0 autumn 2007
spring 2008
in shade leaves of the oldest trees in summer (Chls/ Cars = 12) (Fig. 5c2). The amount of total soluble proteins per unit leaf area was higher in sun than in shade leaves in all trees (Fig. 6a1, b1, c1), although the difference was significant only in the 13-year-old trees in summer (Fig. 6b1). In addition, protein contents increased in both sun and shade leaves from spring to summer, the highest increases corresponding to sun leaves of the 13-year-old (Fig. 6b1) and the oldest trees (Fig. 6c1). However, the greatest total Rubisco activity in spring and summer was measured in sun leaves of the youngest trees (around 15 lmol CO2 m-2 s-1) (Fig. 6a2), being these values two or three times higher than those measured in sun leaves of the 13-year-old (Fig. 6b2) and the oldest trees (Fig. 6c2) in spring and summer. Except for the oldest trees in summer (Fig. 6c2), Rubisco activity was always lower in shade than in sun leaves regardless of tree age and leaf phenology (Fig. 6a2, b2, c2). Sun leaves accumulated greater content of total soluble sugars (TSS) than shade leaves in spring, regardless of tree age (Fig. 7a1, b1, c1). In the oldest trees, sun leaves also showed higher TSS levels than shade leaves in summer
123
summer 2008
autumn 2007
spring 2008
summer 2008
(Fig. 7c1). The lowest amount of TSS per unit leaf area in both sun and shade leaves of all trees was measured in autumn. The presence of TSS in leaves was negatively correlated to starch content. In fact, the greatest accumulation of starch occurred in both sun and shade leaves in autumn (Fig. 7a2, b2, c2), being levels greater in sun than in shade leaves independent of tree age. Water content in sun and shade leaves of the youngest trees was comparable in autumn and spring and it was slightly higher in shade than in sun leaves in summer (Fig. 8a1). In contrast, shade leaves of both 13-year-old and the oldest trees always had greater water content than sun leaves, being such difference more evident in spring (Fig. 8b1, b2). The fact that leaf relative water content (RWC) always reached values that exceeded 86% revealed that both sun and shade leaves exhibited optimal water status independent of tree age and season of the year (Fig. 8a2, b2, c2). However, RWC decreased in sun leaves of the youngest (Fig. 8a2) and the oldest trees (Fig. 8c2) from spring to summer. The decline of RWC in shade leaves of these trees from spring to summer was very slight (Fig. 8a2, c2).
Trees (2010) 24:1029–1043 4
Sun Shade b c
3
16
a1
a
a2 14
b
b
12
c
10
a
2
b
bc
6
cd
d
1
8
a
4 2 0
0
b1
a b
Chl a/b C
3
b2 14
bc
cd
d
12
a
e
2
b
8
b
6
c
1
10
a
c
Chls/Cars
Fig. 5 Ratios of pigments, Chl a/b (a1, b1, c1) and total chlorophylls (a ? b) to total carotenoids (x ? c) (Chls/Cars) (a2, b2, c2) in sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clear-cut in 2001 (a1, a2), the stand clearcut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05). Chl chlorophyll, Cars total carotenoids, x xanthophylls, c carotenes
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4 2 0
0
c1 a a
a
3
c2 14
a
12
a
a 2
bc
b
10
b
8
bc
bc
6
c
1
4 2
0
spring 2008
autumn 2007
summer 2008
0.25
0.15
Total soluble proteins (mg cm-2)
ab
abc
0.05
a
10
b
5
b
0
a
b1
0.20
b2
a b
0.10
b b
b
b
b
10
b b
0
c1
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b
5 0
a
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a 0.15 0.10
b ab
ab b
b
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d
cd cd
0 autumn 2007
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20 15
c
b 0.05
20 15
b
0.05
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b
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c
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15
abc
0
0.15
a2 a
a
0
summer 2008
a1
0.20
0.10
spring 2008
Rubisco activity (µmol CO2 m-2 s-1)
Fig. 6 Total soluble proteins (mg cm-2) (a1, b1, c1) and Rubisco activity (lmol m-2 s-1) (a2, b2, c2) in sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clear-cut in 2001 (a1, a2), the stand clear-cut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05)
autumn 2007
spring 2008
summer 2008
5 0
123
1038 3.5
a1
3.0
0.20
2.0
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05.
d
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0
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c c
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spring 2008
Transpiration decline curves (Fig. 9) revealed clear differences between sun and shade leaves, but only in spring. When working with fully expanded young leaves, it was possible to infer the leaf RWC value at which stomata closure occurred because there was an inflection point between the exponential and the linear phases of leaf water loss. According to the inferred inflection points, we can affirm that stomata closure in sun leaves took place at higher values of RWC than in shade leaves. In sun leaves collected in spring, stomata closure occurred at RWC of 75–78% regardless of tree age. On the other hand, in shade leaves collected in spring, stomata closure took place at RWC varying from 55% in the 13-year-old trees (Fig. 9b2) to near 70% in the youngest (Fig. 9a2) and the oldest (Fig. 9c2) beech trees.
Discussion Leaf area index (LAI, the total one-sided foliage area per unit ground surface area) controls light interception of plant canopies, and influences carbon and water exchange between vegetation and the atmosphere (Leuchner et al. 2006). Stand leaf area, which depends on both leaf size and the total number of leaves per plant, is not the only
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0.05 0
a
a
2.5
0.5
0.20
b ab
0.5
0.05 0
a
a
2.0
1.0
a2
a
2.5
1.0
0.25
a
Starch (mg cm-2)
Fig. 7 Total soluble sugars (TSS) (mg cm-2) (a1, b1, c1) and starch (mg cm-2) (a2, b2, c2) contents in sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clearcut in 2001 (a1, a2), the stand clear-cut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05)
Trees (2010) 24:1029–1043
summer 2008
autumn 2007
spring 2008
summer 2008
structural parameter that can determine LAI. In fact, stem density may influence LAI (Le Dantec et al. 2000; Leuchner et al. 2006). In our study, the lowest values of LAI were measured within the stand clear-cut in 2001, which could be due to the smaller size of the youngest trees and, consequently, their smaller number of leaves and thus their presumably smaller total leaf area per plant. In contrast, the highest LAI values across the year corresponded to the stand disturbed in 1996, which indicates the relevant influence of stem density on LAI. Stem density is usually negatively correlated with stand age (Leuchner et al. 2006), but this is not the case in our study because the stand clearcut in 1996 had greater stem density than that disturbed in 2001. Values of SLA of sun and shade leaves in our study were similar to those found by Sarijeva et al. (2007) and Lichtenthaler et al. (2007) for F. sylvatica. The greatest SLA always corresponded to shade leaves, which means that those leaves increased the efficiency of light interception by maximizing the leaf area per unit leaf biomass (Sarijeva et al. 2007; Petritan et al. 2009). Several authors (King 2003; Barthod and Epron 2005; Petritan et al. 2009) have found that SLA increase is directly related to the shade tolerance rank of tree species. Beech is considered to be one of the most shade-tolerant tree species in Europe (Ellenberg
Trees (2010) 24:1029–1043 3.5 3.0
Sun Shade
a1
ab
a abc
2.0
bc
ab
ab
100 95
bc
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1.5
a2 c c
a
b
90
c
1.0
85
0.5
WC (g H2O g-1 DM)
0
a
b1
3.0
80
a ab
2.5 2.0
ab
b
ab
95
bc
cd
cd
ab
b2
b 90
d
1.5 1.0
RWC (%)
Fig. 8 Water content (WC) (g H2O g-1 leaf DM) (a1, b1, c1) and relative water content (RWC) (%) (a2, b2, c2) in sun (white histograms) and shade (black histograms) leaves of beech trees from the stand clearcut in 2001 (a1, a2), the stand clear-cut in 1996 (b1, b2) and the unmanaged forest (c1, c2). Leaves collected on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Histograms represent means (n = 45) ± SE. Within each graph, histograms with the same letter indicate that values did not differ significantly between sun and shade leaves across the seasons (p B 0.05). DM dry matter
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85
0.5 0
80
c1
3.0
a
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b
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c
a
c2 ab
b
c
ab
95
b b b
d
1.0
90 85
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autumn 2007
spring 2008
1988) and our results show that SLA adaptation of shade leaves of beech can vary across the seasons of the year. Stomata index in sun leaves was quite similar among trees of different age throughout the annual cycle. In contrast, stomata index in shade leaves seemed to be negatively correlated to tree age. While stomata index was comparable for sun and shade leaves in the youngest trees, it was around 35% lower in shade than in sun leaves in the 13-year-old trees and 50% smaller in shade than in sun leaves in the oldest trees. Since PPFD values were different among beech stands, the concept of ‘‘shade’’ leaves also differed among the three studied ecosystems and, consequently, stomata index or stomata initiation could be also determined by light regime. However, in spring, the season when leaves are developing, the highest attenuation of light occurred within the stand clear-cut in 1996 but the greatest difference in stomata index between sun and shade leaves corresponded to the oldest trees. This fact shows that tree age was the more determining factor for stomata index than light. Stomata density was higher in sun than in shade leaves regardless of tree age and leaf phenology, which agrees with observations of Lichtenthaler (1981). In spring, the youngest trees had sun and shade leaves of similar size and
summer 2008
autumn 2007
spring 2008
summer 2008
80
comparable stomata index. Therefore, the different stomata density indicates that sun and shade leaves had different number of epidermal cells on the leaf surface (Hovenden and Vander Schoor 2003). The higher stomata density of sun leaves means that the total number of epidermal cells was increased (Hovenden and Vander Schoor 2003). In general, it is known that cell division in plants is correlated with carbohydrate supply, and that leaves starved of photosynthate or other nutrients will develop fewer cells than those growing amid plenty (Dale 1988). In our study, shade leaves contained lower amount of TSS than sun leaves in spring, but not only in the youngest trees. Both the 13-yearold and the oldest beech trees also had higher content of TSS in sun than in shade leaves in spring. Therefore, carbohydrate supply would not explain differences in the number of epidermal cells in shade leaves of beech trees differing in age. Differences in the levels of Chl a ? b per leaf area between sun and shade leaves strongly depended on tree age and season of the year. According to Hansen et al. (2002), the leaf area-based Chl a ? b content is affected by a multiplicity of factors such as the leaf structure-dependent light gradient within the leaf and the resulting ratio of sun-type chloroplast to thylakoid and Chl-enriched shade-
123
123
Measurements taken on 25 October 2007 (autumn), 28 May 2008 (spring), and 3 September 2008 (summer). Values represent means (n = 10) ± SE. Within each parameter and beech stand, values followed by the same letter do not differ significantly across the seasons (p B 0.05)
19.8 ± 0.1 a 11.7 ± 0.1 b 4.5 ± 0.0 c 16.4 ± 0.2 a 9.8 ± 0.2 b 12.8 ± 0.0 b
19.6 ± 0.1 a 4.0 ± 0.0 c Air temperature inside (°C)
13.0 ± 0.0 b
0.9 ± 0.1 a
20.4 ± 0.3 a 4.2 ± 0.0 c
3.6 ± 0.03 c
2.2 ± 0.3 b
20.3 ± 0.3 a 12.0 ± 0.0 b 4.9 ± 0.1 c 17.0 ± 0.0 a 10.0 ± 0.1 b
14.6 ± 2.1 c
Air temperature outside (°C)
3.7 ± 0.10 c
3.9 ± 0.2 a
27.5 ± 3.9 c 5.0 ± 0.9 c
1.0 ± 0.3 c 4.2 ± 0.2 b
68.0 ± 11.7 c 6.4 ± 1.1 d
6.6 ± 0.2 a 1.6 ± 0.1 c
6.3 ± 0.8 d
LAI (cm2 cm-2)
0.7 ± 0.1 a
6.0 ± 0.4 f 160.0 ± 8.0 d 239.4 ± 23.7 c
0.1 ± 0.0 b
PPFD Shade leaves (lmol m-2 s-1)
15.7 ± 0.3 a 10.7 ± 0.1 b
33.0 ± 5.3 c 678.4 ± 63.9 a 462.4 ± 12.6 b
9.2 ± 0.2 c 14.5 ± 0.2 a 9.3 ± 0.2 b 8.7 ± 0.1 c
41.1 ± 3.7 e 339.9 ± 9.4 b 415.7 ± 7.7 a
15.9 ± 0.3 a 11.2 ± 0.3 b 10.3 ± 0.1 c Soil temperature (°C)
PPFD Sun leaves (lmol m-2 s-1)
32.9 ± 1.5 cd 1,374.8 ± 34.3 a 370.4 ± 5.6 b
26.3 ± 1.6 c 57.2 ± 0.2 a 41.9 ± 1.1 b 55.4 ± 1.0 a 65.5 ± 1.7 a 60.7 ± 4.8 a 31.8 ± 2.4 b 40.7 ± 0.5 a
Spring
30.9 ± 1.5 b Soil water content (g H2O g-1 dried soil 9 100)
Autumn Autumn Autumn
Summer
Clear-cut in 1996
Spring
Summer
Unmanaged
Spring
Summer
Trees (2010) 24:1029–1043
Clear-cut in 2001
Table 2 Soil water content, soil temperature, photosynthetic photon flux density (PPFD) on sun and shade leaves, leaf area index (LAI) and air temperature outside and inside the beech stands clear-cut in 2001 or 1996 and within the unmanaged forest
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type chloroplasts. When Chl a/b ratio in sun leaves is higher than in shade leaves, lower Chl a ? b content in sun leaves can be due to the greater proportion of sun-type chloroplasts and photosynthetic units with sun-type characteristics (Hansen et al. 2002). In our study, this explanation can be applied to the youngest trees in spring and to both the 13-year-old and the oldest beech trees in summer. On the other hand, in the oldest beech trees, the highest content of Chl a ? b in spring corresponded to sun leaves, which agrees with results obtained by some authors (Lichtenthaler et al. 1981; Sarijeva et al. 2007). Garcı´a-Plazaola and Becerril (2001) concluded that decline of total Chls and thus leaf senescence in beech occurred earlier in sun than in shade leaves. In our study, however, only in the oldest trees the levels of Chls clearly decreased earlier in sun than in shade leaves. In agreement with several authors (Lichtenthaler et al. 2007; Sarijeva et al. 2007), the amount of total Cars in sun leaves was greater than those in shade leaves in summer. However, we observed a seasonal pattern related to Cars levels in sun and shade leaves that depended on tree age. In the youngest trees, the content of Cars was higher in shade than in sun leaves when leaves were still developing (spring). The organization of the pigment apparatus and the relative levels of Chl a and Chl b as well as the ratio of total Chl a ? b to total Cars are essential differences between sun and shade leaves (Lichtenthaler et al. 2007). In our study, in general terms, shade leaves possessed lower Chl a/b ratios, which agrees with several authors working with beech (e.g., Sarijeva et al. 2007; Lichtenthaler et al. 2007). However, there was an exception: sun and shade leaves of the oldest beech trees had comparable Chl a/b ratios in autumn and spring, coinciding, respectively, with senescence and early development of leaves. As PPFD was very different for sun and shade leaves in spring, we can conclude that phenological stage of leaves strongly influenced pigment composition in the oldest beech trees. Similarly to Chl a/b ratios, the ratio of total Chls to total Cars was lower in sun than in shade leaves except for the similar values observed in both types of leaves in autumn and spring for the oldest beech trees. Therefore, our results suggest an important influence of tree age on the structural development and physiology of sun and shade leaves. High stomata density together with great stomata conductance seems to be a typical characteristic of sun leaves and a prerequisite for their greater level of photosynthesis (Lichtenthaler and Babani 2004). In our study, only in the youngest trees, total Rubisco activity was significantly higher in sun than in shade leaves throughout the annual cycle. In contrast, Herbinger et al. (2005) found that decline in photosynthesis from sun-exposed to shaded conditions was more pronounced in adult than in juvenile beech trees. Moreover, in spring and summer, sun leaves of the youngest
Trees (2010) 24:1029–1043
30 25
spring 2008
a2
a3
summer 2008 30 Sun leaves 25 Shade leaves
20
20
15
15
10
10
5
5
0
mg H2O m-2 s-1
autumn 2007
a1
25
b1
b2
0
b3
25
20
20
15
15
10
10
5
5
0 25
c1
c2
0 25
c3
20
20
15
15
10
10
5 0 100
80
60
40
20
0
80
60
40
20
0
mg H2O m-2 s-1
Fig. 9 Transpiration decline curves (TDC) (water loss vs. relative water content, RWC) corresponding to sun (white symbols) and shade (black symbols) leaves of beech trees from the stand clear-cut in 2001 (a1, a2, a3), the stand clear-cut in 1996 (b1, b2, b3) and the unmanaged forest (c1, c2, c3). TDC carried out on leaves collected on 25 October 2007 (autumn) (a1, b1, c1), 28 May 2008 (spring) (a2, b2, c2), and 3 September 2008 (summer) (a3, b3, c3). Each symbol represents means (n = 45)
1041
80
60
40
20
5 0 0
RWC
beech trees exhibited the greatest total Rubisco activity. However, the content of TSS in sun leaves was similar among beech trees regardless of tree age. Therefore, it is possible that recently synthesized sugars by photosynthesis in young trees will be quickly transported from leaves to roots, stem, and branches actively growing. Although young twigs are known to be photosynthetically active organs able to contribute substantially to the overall carbon budget and carbon balance of woody plants, including F. sylvatica (Wittmann et al. 2001), twigs are more costly with respect to carbon and energy than leaves during their development, because twigs require larger investments of resources for their construction (Comstock and Ehleringer 1988). In older beech trees, even 100–150-year-old stems still possess a chlorophyll-containing chlorenchyma right below their very thin peridermal layers so that the longer lifetime of the twigs could thus positively contribute to partly compensate the higher construction and maintenance costs of twigs as compared to their short-lived leaves (Wittmann et al. 2001). Consequently, the transport of sugars from leaves to roots and stems will presumably be slower in older beech trees than in young individuals. Moreover, an additional sink of leaf carbohydrates will be ectomycorrhizal fungi (ECM) associated with beech roots. Goicoechea et al. (2009) studying ECM diversity, within the same beech stands described in this paper, observed a significant increase in the percentage of ECM colonization in the youngest beech trees (those growing within the stand clear-cut in 2001) from winter to spring while the percentage of roots associated with ECM remained almost
constant in older trees from the cold to the warm season or even decreased in the 13-year-old trees in autumn. According to Last et al. (1979) most part of ECM fungi seems to rely on supplies of recent photosynthate that is sugars produced in leaves. In addition, the presence of ECM in beech roots could have contributed to enhance photophosphorylation rates (Smith and Gianinazzi-Pearson 1988), so that the increased ECM colonization observed in the youngest trees in spring (Goicoechea et al. 2009) could be related to their higher Rubisco activity. When compared to the levels of non-structural carbohydrates (TSS and starch) between sun and shade leaves, we always found higher levels in sun than in shade leaves. Only in sun leaves PPFD strongly affected the contents of both TSS (r = 0.565, p B 0.05 in the stand clear-cut in 2001, r = 0.733, p B 0.01 in the stand clear-cut in 1996 and r = 0.842, p B 0.01 in the unmanaged stand) and starch (r = -0.961, p B 0.001 in the stands clear-cut in 2001 or 1996 and r = -0.699, p B 0.01 in the unmanaged stand). When compared to the contents of non-structural carbohydrates in shade leaves of different beech trees, we observed that beech trees growing within the stand clear-cut in 1996 (where the strongest light attenuation occurred) had similar or slightly lower levels of TSS and starch than beech trees within the stand disturbed in 2001 and the unmanaged forest. Recently, Piper et al. (2009), studying the carbohydrate storage of two Nothofagus species in two contrasting light environments concluded that the concentration of nonstructural sugars was influenced by light environment but survival in deep shade was not correlated with the
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concentration of non-structural sugars since leaves of Nothofagus always showed higher contents of non-structural sugars under light than under deep shade conditions. Similarly, according to findings of Lichtenthaler et al. (2007), shade leaves had greater water content than sun leaves but only within the ecosystems showing the strongest light differences between sun and shade conditions (the stand clear-cut in 1996 and the unmanaged forest). In fact, the highest difference between shade and sun leaves was observed in beech trees growing within the stand disturbed in 1996 in spring, coinciding with the lowest PPFD and the highest soil moisture inside this ecosystem. There was a positive correlation between the soil moisture and the leaf water content of both sun (r = 0.664, p B 0.01) and shade (r = 0.661, p B 0.01) leaves of the 13-year-old beech trees. However, when the water status of leaves was calculated taking into account the maximum amount of water that leaves can contain turgid weight, we observed that the leaf RWC was more affected by the microclimatic conditions in both the youngest and the oldest trees than in the 13-yearold beech trees. The decreased RWC in sun leaves of the youngest trees in summer could be due to the increased PPFD and the declined soil water content. In fact, Pearson’s correlation analyses showed that RWC was negatively affected by PPFD (r = -0.515, p B 0.05) and positively related to soil moisture (r = 0.824, p B 0.001) within the stand clear-cut in 2001. In the oldest trees, the decrease in the RWC of sun leaves would be a consequence of decreased soil moisture since both parameters were significantly correlated (r = 0.688, p B 0.01). When representing water loss versus RWC in leaves we found that stomata closure in young leaves, in spring, always occurred at higher RWC in sun than in shade leaves, which indicates enhanced stomata sensitivity in sun leaves. Leaf stomata sensitivity can be affected by PPFD and leaf-to-air vapor deficit (VPD) (Kutsch et al. 2001) and can be modified by acclimation to soil water status (Halldin et al. 1984). However, in contrast to our results, Kutsch et al. (2001) concluded that reduction in stomata aperture of beech leaves in dry air was negatively correlated with the sum of PPFD of the previous 3 weeks. The behavior observed for stomata closure in young leaves was not clearly observed in leaves collected in summer and autumn presumably because the thicker cuticle of older leaves would have affected total leaf conductance to a greater extent than in young leaves. Moreover, phenology can also influence leaf stomata sensitivity (von Stamm 1994).
Conclusion PPFD, air temperature, soil moisture, and soil temperature within beech stands affected the physiology and
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Trees (2010) 24:1029–1043
biochemistry of both sun and shade leaves being the morphology and anatomy of leaves probably more correlated to the environmental conditions, which occurred during the period of leaf formation. The extent to what those microclimatic parameters affected leaf physiology seemed to be dependent on age of beech trees, being both sun and shade leaves of the oldest trees the most influenced by PPFD, air temperature, soil water content and soil temperature. Acknowledgments Our research was supported by Fundacio´n Universitaria de Navarra (FUNA, PIUNA) and Caja Navarra. Iva´n Closa was a recipient of a grant from Asociacio´n de Amigos de la Universidad de Navarra (ADA). The authors are grateful to Lorenzo Etxarri for assistance with logistics, site locations and history information on forest management as well as Amadeo Urdiain for technical assistance.
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