Tree Physiology 33, 730–742
doi:10.1093/treephys/tpt045
Research paper
Seasonal and inter-annual dynamics of growth, non-structural
carbohydrates and C stable isotopes in a Mediterranean beech
forest
Andrea Scartazza1,2,5, Stefano Moscatello2, Giorgio Matteucci1,3, Alberto Battistelli2 and
Enrico Brugnoli2,4
1Istituto
di Biologia Agroambientale e Forestale (IBAF), Consiglio Nazionale delle Ricerche (CNR), Via Salaria km 29,300, 00016 Monterotondo Scalo (RM), Italy; 2Istituto di
Biologia Agroambientale e Forestale (IBAF), Consiglio Nazionale delle Ricerche (CNR), Viale G. Marconi 2, 05010 Porano (TR), Italy; 3Istituto per i Sistemi Agricoli e Forestali
del Mediterraneo (ISAFoM), Consiglio Nazionale delle Ricerche (CNR), Via Cavour 4/6, 87036 Rende (CS), Italy; 4Present address: Dipartimento Scienze del Sistema Terra e
Tecnologie per l’Ambiente, Consiglio Nazionale delle Ricerche (CNR), Piazzale Aldo Moro 7, 00185 Roma (RM), Italy; 5Corresponding author ([email protected])
Received February 19, 2013; accepted May 27, 2013; published online July 11, 2013; handling Editor Peter Millard
Seasonal and inter-annual dynamics of growth, non-structural carbohydrates (NSC) and carbon isotope composition (δ13C) of
NSC were studied in a beech forest of Central Italy over a 2-year period characterized by different environmental conditions.
The net C assimilated by forest trees was mainly used to sustain growth early in the season and to accumulate storage carbohydrates in trunk and root wood in the later part of the season, before leaf shedding. Growth and NSC concentration
dynamics were only slightly affected by the reduced soil water content (SWC) during the drier year. Conversely, the carbon
isotope analysis on NSC revealed seasonal and inter-annual variations of photosynthetic and post-carboxylation fractionation
processes, with a significant increase in δ13C of wood and leaf soluble sugars in the drier summer year than in the wetter one.
The highly significant correlation between δ13C of leaf soluble sugars and SWC suggests a decrease of the canopy C isotope
discrimination and, hence, an increased water-use efficiency with decreasing soil water availability. This may be a relevant
trait for maintaining an acceptable plant water status and a relatively high C sink capacity during dry seasonal periods. Our
results suggest a short- to medium-term homeostatic response of the Collelongo beech stand to variations in water availability and solar radiation, indicating that this Mediterranean forest was able to adjust carbon–water balance in order to
prevent C depletion and to sustain plant growth and reserve accumulation during relatively dry seasons.
Keywords: carbon isotope composition, carbon reserves, Fagus sylvatica, water stress, water-use efficiency.
Introduction
Photosynthetic products can be used to sustain plant growth or
stored as non-structural carbohydrates (NSC), or other reserve
compounds, in sink organs (e.g., Ho 1988). In tree species, a
substantial part of the biomass is represented by xylematic tissue, composed by vessel elements or tracheids, fibers and parenchymatic cells. These cells, in the sap wood, are able to
accumulate consistent amounts of reserves, mainly NSC and
lipids (Kramer and Kozlowski 1979). Partitioning and accumulation of carbohydrate reserves among plant organs and tissues
are affected by several factors, including temperature (Sauter
1988), light (Gansert and Sprick 1998), drought (McDowell
et al. 2008, Woodruff and Meinzer 2011), ozone (Landolt et al.
1994, Battistelli et al. 2001), altitude (Bernoulli and Körner
1999), tree size (Woodruff and Meinzer 2011) and defoliation
(Cerasoli et al. 2004). Wood carbohydrates play a key role in
allowing plant survival during adverse conditions (e.g., Ögren
© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Growth, non-structural carbohydrates and δ13C dynamics in beech 731
2000, Barbaroux et al. 2003). For example, during drought
periods with reduced photosynthetic capacity and transpiration,
many tree species mobilize the storage carbohydrates to satisfy
the continued metabolic demand (Ryan 2011). Hence, drought
generally causes a decrease in reserve carbohydrates of plants
(Sala et al. 2010) leading to C depletion and eventually increasing tree mortality (McDowell et al. 2008). Furthermore, a
depletion of carbohydrate reserves can progressively decrease
C allocation to defense compounds, reducing plant resilience to
other disturbance factors, such as insect attack and diseases
(Allen et al. 2010, van der Molen et al. 2011). However, Silpi
et al. (2007) suggested that carbohydrate reserves in trees are
not a mere passive buffer, but rather trees tend to adjust the
amount of reserves stored to the level of metabolic demand at
the possible expense of growth, therefore acting as competing
C sinks. Galvez et al. (2011) found a drought-induced increase
of root carbohydrate reserves in aspen seedlings, while Piper
(2011) showed that drought-induced mobilization of C storage
in two evergreen Nothofagus species is affected by drought
intensity and by drought resistance of the species, with a
decrease of NSC content in the more drought-susceptible species and an increase in the relatively more drought-resistant
ones. It has been demonstrated in several deciduous species
that reserves are mobilized at bud break to sustain spring
growth (e.g., Gregory and Wargo 1986, Wong et al. 2003) and
that a severe drought period affecting reserve accumulation
and partitioning may also influence phenology and spring
growth rate in the subsequent year (Scartazza et al. 2001,
Bréda et al. 2006). Reserves are also mobilized during winter in
deciduous species for acclimation to low temperatures, development and maintenance of cold tolerance and cellular respiration (e.g., Ögren 2000, Barbaroux et al. 2003). Elevated levels
of sucrose, glucose and fructose during the cold season have
been observed in drought-stressed maple trees, suggesting a
potential role of these carbohydrates as osmoregulators for
freeze protection (Wong et al. 2009).
The isotopic analysis of NSC is a powerful tool to obtain information on C and water relations at plant and ecosystem level
(for some reviews see Brugnoli and Farquhar 2000, Brugnoli
et al. 2012, Werner et al. 2012). Carbon isotope signature of
soluble sugars and starch is dependent on the photosynthetic C
isotope discrimination (Δ, Brugnoli et al. 1988). This physiological parameter is linearly related to the ratio of intercellular and
atmospheric CO2 partial pressure (pi/pa), and hence it is
inversely related to water-use efficiency (WUE, Farquhar et al.
1989). Water deficit and other environmental drivers cause
large variations of canopy Δ, and hence of carbon isotope composition (δ13C) of sugars assimilated by leaves and transported
towards different sink organs. The analysis of δ13C on NSC in
different above- and below-ground plant components has been
previously used to study the effects of drought on partitioning
of photosynthetic assimilates among plant organs during the
growing season in walnut plants (Scartazza et al. 2001) and to
study the seasonal variations of sugar δ13C in leaves and stems
of beech plants (Damesin and Lelarge 2003).
Plants are composed of different types of source and sink tissues; hence, the evaluation of the whole tree carbon balance is
affected by the source–sink relationships and the interaction of all
tissues among themselves and with the surrounding environment.
Consequently, the analysis of only a part of the plant body and of
the effects of a single environmental factor is not sufficient to
understand the whole system and the source–sink relationships at
the whole plant level. It has been highlighted that measurements
of plant C balance are relatively simple to do for seedlings and
saplings and in pot experiments, but still represent a challenge for
large trees and in natural forest environments (Ryan 2011). Hence,
a whole tree approach in natural environments, taking into account
different plant organs and tissues and environmental drivers, is
necessary to understand how drought or other environmental and
phenological factors affect physiological performances and plant
C balance in forest ecosystems. Beech (Fagus sylvatica L.) represents one of the most important European forest tree species
(Tarp et al. 2000). Hence, any possible adverse effects on this
species can have great ecological and economic impacts in
Europe (Peuke et al. 2002, Gessler et al. 2007). In particular,
physiological performances, growth and competitive ability of
European beech may be adversely affected by low water availability and long drought periods (e.g., Fotelli et al. 2002, Gessler
et al. 2004). During the severe summer drought in 2003, beech
forests of Central Europe were among those which showed the
largest reductions in net ecosystem productivity (Ciais et al.
2005). However, it has been suggested that European beech of
southern provenances might be able to cope better with drought
events than beech of Central European origins (Peuke et al.
2002), indicating genetic specialization involving the presence of
efficient internal regulation mechanisms under the relatively dry
Mediterranean climate (Nahm et al. 2006).
In the present study, the seasonal and inter-annual variability
of growth, carbohydrate reserves and δ13C of NSC in wood and
leaves of a beech forest in the Central Italy during 2 years characterized by contrasting summer water availability were analyzed. The main objectives of this work were (i) to determine
the role of phenology and climate on the control of storage
carbohydrate accumulation and growth dynamics; (ii) to identify
possible causes of seasonal and inter-annual variations in δ13C
in NSC of source and sink tissues; and (iii) to evaluate the role
of WUE on C sink and partitioning during relatively dry periods.
Materials and methods
Experimental site
The study was carried out in a beech (Fagus sylvatica L.) forest
near Collelongo (Abruzzo region, Central Italy). A permanent
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732 Scartazza et al.
experimental facility (Selva Piana stand, 41°50′58″ N,
13°35′17″ E, 1550 m elevation) was installed in 1991 by the
Department of Forest Environment and Resources, University of
Tuscia, Viterbo, Italy. Since 2004, the facility has been run by
the Institute of Agro-Environmental and Forest Biology of the
National Research Council of Italy. The site is a part of several
international networks (CarboEurope, FluxNet, ICP-Forests) and
since 2006 has been one of the sites of the Italian Long Term
Ecological Research network (Cocciufa et al. 2011). The Selva
Piana stand is located within a 3000-ha community forest that
is part of a wider forest area, included in the external belt of the
Abruzzo National Park. The environmental and structural conditions of the stand are representative of Central Apennine beech
forests. In the area of the experimental site, the density is
830 trees ha−1, the average basal area is 39.4 m2 ha−1 with a
mean diameter at breast height of 24.6 cm (sampling trees with
diameter >1 cm) and a mean height of 21.2 m (data from the
2002 survey). Over a 10-year period, stand leaf area index
(LAI) ranged between 4.5 and 6.5 (D’Andrea, 2005). Mean
tree age has been measured to be around 100 years in 1996.
The soil, developed on a calcareous bedrock, has a variable
depth (40–100 cm) and can be classified as a humic alisol. Site
topography is gently sloped. The mean annual temperature and
the annual precipitation for the period 1996–2009 are 7.08 °C
and 1088 mm year−1, respectively. Air temperature and rainfall
during 2001 and 2002 are shown in Figure 1.
The site is equipped with a 26-m high scaffold tower with an
additional mast reaching the height of 32 m, ~8–10 m above the
canopy. Meteorological sensors are available on the tower at different heights along the canopy profile as previously described
(Matteucci et al. 2007). Air temperature, air humidity and wind
speed sensors were mounted at different heights. Incoming total
and diffuse photosynthetically active radiation (PAR; Li190,
LiCor, Lincoln, NE, USA and SKYE, Powys, UK) and net radiation
(REBS, Seattle, WA, USA) were measured above the top of the
canopy. Rainfall was measured above (27.4 m) and below the
canopy (1.5 m) by rain gauges. Soil temperature was measured
at two depths (0.05 and 0.20 m). All meteorological data were
recorded by two Campbell CR10 data loggers (Campbell, Logan,
UT, USA). Ecosystem water and CO2 fluxes have been measured
since 1993 with eddy covariance technique, using the
EUROFLUX setup as previously described (Aubinet et al. 2000).
The experimental setup consisted of a fast response infrared
gas analyzer (LI-6262, LI-COR, Lincoln, NE, USA) and a threedimensional sonic anemometer (SOLENT 1012R2, Gill
Instruments, Lymington, UK). Air was drawn through the analyzer by a pump installed downstream of the analyzer. In terms
of flux-footprint area, the study-site provided a large fetch of
~2.5 × 2 km2 (NNE-SSW× WNW-ESE) over a continuous and
homogenous beech forest.
Soil water content measurements
Soil water content (SWC) was measured with a portable TDR
(Time Domain Reflectometry) instrument (Trime, IMKO GmbH,
Ettlingen, Germany). Five permanent 0–100 cm especially
designed plastic tubes (TECANAT, IMKO GmbH) were used.
Measurements were taken twice monthly using a 18-cm-long
probe (TRIME-T3 probe). Five measurements were taken for each
of four different profiles, approximately at 0–18, 32–50, 52–70
and 70–88 cm depths. In Figure 2a, the average value of SWC
determined between 0 and 88 cm depth is reported. The record
of August 2002 is missing due to an instrumental problem.
Stem growth measurements
Stem growth was measured with dendrometer bands
(DIALDENDRO, FOB GmbH, Salzburg, Austria) installed on
Figure 1. ​Thermopluviometric diagram of the Collelongo site for the years 2001 and 2002. Data of air temperature and rainfall are presented,
respectively, as the average and the sum of ten days.
Tree Physiology Volume 33, 2013
Growth, non-structural carbohydrates and δ13C dynamics in beech 733
Wood cylinders of ~5 mm diameter and 3 cm length were collected at the trunk base (~50 cm from the soil) of 10 plants in
2001 and 2002 and from the coarse roots of 6 plants (only
from July to November 2002). Leaves were collected in
September of both years from three canopy heights (~24, 19
and 13.5 m) at random position within the canopy before dusk.
Wood and leaf samples were immediately frozen in liquid N2,
then ground to a fine powder and, finally, used for starch and
soluble sugar extraction. An aliquot of the leaf and wood powder (~100 mg) was used to extract starch and soluble sugars
for δ13C analysis, while another aliquot (~10 mg) was used to
extract starch and soluble sugars for the carbohydrate quantitative analysis. Stem bark sugars were collected after leaf
shedding in November from five (2001) and eight (2002)
trees as described in detail by Scartazza et al. (2004). Briefly,
a small rectangle of stem bark (~2 × 4 cm) was cut at ~1.3 m
from the ground leaving the upper horizontal side attached to
the trunk. This bark–phloem portion was separated by cambium and, after thoroughly washing with distilled water, its
basal edge was placed into a plastic tube containing an EDTA
solution (10 mM) to collect sugars throughout the daily light
period (~8–10 h). After dusk, tubes were removed, placed in a
cooler at 0 °C and transported to the laboratory where sugars
were purified. EDTA and other ions were removed by using ion
exchange resins, as in Scartazza et al. 2004. In November, the
canopy photosynthesis was absent; hence, during the relatively
long collection period, we likely sampled sugars present in
phloem and sugars of the apoplast that could diffuse from the
tissue surrounding the cut surface to the collection tube. We
cannot quantify the relative contribution of phloem and apoplast sugars to the collected amount; hence, in this work, we
indicated these soluble carbohydrates as ‘bark sugars’ instead
of ‘phloem sap sugars’ as used in Scartazza et al. (2004).
Figure 2. ​
Seasonal variations of SWC determined between 0 and
88 cm of depth (a) and of the monthly global solar radiation (b).
Values of SWC are means ± SE (n = 5). Soil water content values for
the year 2001 are from Scartazza et al. (2004). The value of August
2002 is missing due to an instrumental problem.
nine trees representative of the various structural conditions of
the canopy. This setup allowed us to detect circumference
increase as small as 0.1 mm. Readings were usually taken at
biweekly intervals. Daily diameter growth of Figure 3a is
­calculated as the difference in diameter between two consecutive measurements. Growth was assumed to be linear between
measurement pairs.
Sampling of plant components for carbohydrate
contents and isotopic analyses
Plant components were collected on several occasions from
May to November during 2001 and 2002 growing seasons.
Carbohydrate extraction and purification for
carbon isotope analysis
Total soluble sugars and starch were extracted from ~100 mg
of wood samples consistent with Brugnoli et al. (1988). Briefly,
wood samples were suspended in 5 ml of demineralized water
and shaken for 40 min at room temperature. After centrifugation at 12,000 g, the supernatant was purified as reported
below. The pellet was further used for starch extraction. After
washing in 80% (v/v) ethanol to remove possible pigments,
the residue was suspended in 2 ml of HCl 6 N to solubilize
starch. After centrifugation at 12,000 g for 20 min, the supernatant was retained and the residue was extracted a second
time with HCl. Subsequently, the supernatants were placed
together in a same volumetric flask and starch was separated
by adding 80% (v/v) ethanol, waiting overnight at 5 °C and
centrifuging for 20 min at 12,000 g.
Leaf, wood and bark soluble sugars were purified with
Dowex-50 (H+) resin for the separation of amino acids from
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734 Scartazza et al.
Figure 3. ​Seasonal variations in radial growth and total wood NSC content in the trunk during 2001 and 2002 (a). The individual carbohydrate
contents (starch, sucrose, glucose and fructose) are shown in panel b. Trunk radial growth values for the year 2001 are from Scartazza et al.
(2004). Values are means ± SE (n = 9 for trunk radial growth, n = 10 for trunk carbohydrate content).
organic acids and sugars, and Dowex-1 (Cl−) resin for the separation of organic acids from sugars. The sugar-containing
fractions were freeze-dried and stored dry before carbon isotope analysis.
Quantitative analysis of starch and soluble sugars
About 10 mg of leaf and wood powders were treated separately and analyzed for starch and soluble sugars (glucose,
fructose and sucrose) contents as described in Proietti et al.
(2009). Briefly, these carbohydrates were firstly converted to
the units of glucose-6-phosphate and subsequently enzymatically determined measuring the NADH quantity produced in
the reaction between glucose-6-phosphate and NAD+ in the
presence of glucose-6P-dehydrogenase. The spectrophotometric readings were performed with a dual-wavelength mode
(340–405 nm) in an Anthos 2001 plate reader (Anthos Labtec
Instruments, Salzburg, Austria). Canopy-weighted mean values
Tree Physiology Volume 33, 2013
of leaf sugar concentration were calculated weighting the sugar
concentration of each layer on the LAI of each specific layer.
Carbon isotope analysis
Organic samples were quantitatively combusted using an elemental analyser (Model NA 1500, Carlo Erba, Milan, Italy) coupled with a mass spectrometer. The carbon dioxide produced
from the combustion of organic samples was then analyzed
with a dual inlet mass spectrometer (model SIRA II, VG Isotech,
Middlewich, UK). Carbon isotope composition was calculated
consistent with Farquhar et al. (1989) as
δ13C = (Rs / Rstd ) – 1
where Rs and Rstd are the isotope ratio (13C/12C) of samples
and of the international standard Vienna–Pee Dee Belemnite
(VPDB), respectively.
Growth, non-structural carbohydrates and δ13C dynamics in beech 735
Canopy-weighted mean values of leaf soluble sugar δ13C
canopy) were determined weighting the isotopic composition of each layer on the LAI and the sugar concentration of
each specific layer, consistent with Scartazza et al. (2004).
(δ13C
Statistical analysis
Statistical analysis of the differences in NSC concentration and
δ13C in leaves and wood between 2001 and 2002 was performed by one-way analysis of variance. Linear regression
between δ13Ccanopy and SWC was calculated using the least
squares method.
Results
The diagrams showing relevant meteorological parameters at
the Collelongo site for the years 2001 and 2002 are reported
in Figure 1. Seasonal variations of air temperatures and rainfall
showed relevant inter-annual differences: in 2001, low rainfall
and high air temperatures from June to the first half of
September were recorded, whereas in 2002 an initial period
with decrease in precipitations and increase in air temperatures from the second half of June to July was followed by relatively frequent and intense rainfall events during August and
September (Figure 1). The peculiar thermopluviometric trend
in 2002 was reflected in seasonal differences in SWC and in
global solar radiation (Figure 2). In details, the period from the
end of April to July showed similar SWC trends in the 2 years
2001 and 2002, with high SWC values during bud break and
early spring growth followed by sharp decrease in soil water
availability in July (Figure 2a). However, in 2001, the SWC
remained low throughout the August–September period, while
in 2002 SWC increased again in late summer, and in September
reached values similar to those recorded at the onset of the
growing season (Figure 2a). As a consequence, in September,
the largest inter-annual difference in SWC (~30%) was
observed. The global solar radiation obviously increased from
bud break to June–July and then it decreased until leaf shedding (Figure 2b). However, the global radiation in 2002 was
~20% lower than in 2001 due to the higher cloud cover starting from July.
Figure 3 shows the seasonal trends of trunk radial growth
and of NSC content in trunk wood in 2001 and 2002. Beech
plants showed fast increases of the trunk radial growth during
the first part of the growing season (May–July), reaching a
peak of growth rate in July (Figure 3a). Subsequently, the radial
growth rate decreased sharply and linearly in time during
August–September. A second minor peak was observed in
September and in both years. Hence, our data indicate that
~70% of the annual trunk radial growth occurred between bud
break and July both in 2001 and in 2002. The decrease in
trunk radial growth was associated with an increase in total
NSC in wood. The amount of wood carbohydrates reached a
peak in September; subsequently, it decreased again in
November after leaf shedding (Figure 3a). Although seasonal
trends were similar, it is possible to highlight some differences
between 2001 and 2002. While in 2002 the accumulation of
wood carbohydrates started between July and August, in 2001
this process showed a delay, starting later in the season.
However, despite this seasonal delay, the accumulation rate of
wood carbohydrates was much higher in 2001
(~0.201 mg day−1 from 8 of August to 18 of September) compared with 2002 (~0.109 mg day−1 from 23 July to 27
September). Figure 3b shows the seasonal variations of NSC
extracted from woody tissues: starch, sucrose, glucose and
fructose. From bud break (end of April) to the beginning of
June, a decrease in total soluble sugars and a simultaneous
increase in wood starch collected at the trunk base of both
years were observed, although in 2002 the increase in starch
was higher compared with 2001. The decrease of wood soluble sugars during this initial seasonal period was due to a
simultaneous decrease of individual carbohydrates (sucrose,
glucose and fructose). The starch content increased again
together with soluble sugars until September, when it showed
a seasonal peak. The increase of wood soluble sugars during
this period was due to a sharp increase of the sucrose content,
whereas the amount of glucose and fructose showed only a
slight decrease. Conversely, after leaf shedding, a sharp
decrease of starch associated with a strong increase of glucose and fructose was recorded, while the sucrose content
showed only slight variations.
Seasonal variations of δ13C in both wood soluble sugars
and wood starch extracted from the trunk base are reported
in Figure 4. Carbon isotope composition of wood soluble
sugars was generally higher than that of wood starch, except
in November 2002 when the δ13C values of starch and soluble sugars were similar. Moreover, the isotopic signature of
wood starch sampled in November was very similar to the
δ13C value of the bark sugars collected during the same
period in both years. Wood starch δ13C showed only slight
variations between 2001 and 2002 and throughout the
growing seasons. Conversely, δ13C of wood soluble sugars
showed wide seasonal and inter-annual variation, especially
during 2002. In detail, δ13C of wood soluble sugars increased
from the end of April to June in both years. Subsequently,
during 2001, only a slight variation of δ13C until the end of
the vegetative season was observed, while in 2002 a strong
13C depletion (~2‰) was recorded from June to September.
As a consequence, the seasonal trends of wood soluble
sugar δ13C differed in summer between 2001 and 2002,
with δ13C values of ~1.6‰ lower in September 2002 compared with the same month in 2001. After leaf shedding, in
2001 δ13C of wood soluble sugars decreased reaching the
same values observed at bud break, whereas in 2002 it
showed the lowest seasonal value and was not different
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736 Scartazza et al.
Figure 4. ​Seasonal variations of carbon isotope composition in starch and total soluble sugars in trunk wood during 2001 and 2002. Values are
means ± SE (n = 10). Gray triangles indicate δ13C of bark sugars collected in November 2001 and 2002.
from the isotopic signature of starch and phloem sap
sugars.
Non-structural carbohydrates extracted from the coarse
roots were ~40% higher than those extracted from woody
samples collected at the trunk base from July to November
2002 (compare Figure 3a and Figure 5a). Moreover, after leaf
shedding a strong increase of the total soluble sugars, mainly
glucose and fructose, and a decrease of starch (Figure 5a)
was observed, similar to trunk woody tissues (Figure 3a).
Values and seasonal trends of δ13C in starch and soluble sugars extracted from the coarse roots were similar to those
observed for the trunks (Figure 5b), with a decrease of soluble
sugar δ13C from July to September 2002 and a quite stable
seasonal value of the starch isotopic signature.
Carbon isotope composition of the canopy-weighted mean
value of leaf soluble sugars (δ13Ccanopy) is shown in Figure 6a.
The value of δ13Ccanopy showed wide inter-annual and seasonal
variations: in details, δ13Ccanopy increased during both years
from June to July, following the decrease of SWC (Figure 2).
Subsequently, a different seasonal trend of δ13Ccanopy related to
the inter-annual climatic variability was observed. During the
relatively drier 2001, canopy leaf sugars remained 13C-enriched
also during the second part of the vegetative season; on the
contrary, during the relatively wetter 2002, a strong decrease
of δ13Ccanopy from August to September, associated with the
frequent precipitations and the sharp increase of SWC, was
observed. Consequently, δ13Ccanopy was strongly related to the
inter-annual and seasonal variations of SWC for both years
(Figure 7). Conversely, the canopy-weighted mean value of leaf
soluble sugar concentration showed only slight seasonal and
inter-annual variations (Figure 6b), although during the July–
August period it was lower in 2001 than 2002.
Tree Physiology Volume 33, 2013
Figure 5. ​Seasonal variations of starch and soluble sugar content (a)
and carbon isotope composition (b) in wood of coarse roots collected
from July 2002. Values are means ± SE (n = 6). Gray triangle in panel
b indicates δ13C of bark sugars collected in November 2002.
Growth, non-structural carbohydrates and δ13C dynamics in beech 737
Figure 7. ​Relationship between SWC at 0–88 cm depth and δ13C of
canopy-weighted mean value of leaf soluble sugars for both 2001 and
2002 (r = 0.937, P ≤ 0.01). Values are means ± SE (n = 5 for SWC,
n = 3 for δ13Ccanopy). Values for the year 2001 are from Scartazza et al.
(2004).
Figure 6. ​Seasonal variations of the canopy-weighted mean value of
both leaf soluble sugar δ13C (a) and leaf soluble sugar concentration
(b) in 2001 and 2002. Values are means ± SE (n = 3). Values for the
year 2001 are from Scartazza et al. (2004).
Discussion
Seasonal and inter-annual variability of growth and
storage carbohydrates
In both years, Collelongo beech forest was a C sink from May
to the end of September (Scartazza et al. 2004, data not
shown). The C fixed by the forest from May to July was largely
utilized for plant growth, as indicated by the fast increase of
the trunk radial growth during this period (Figure 3a).
Subsequently, during August and September, the radial growth
drastically decreased, although the forest was still an active
sink for CO2 (Scartazza et al. 2004). At the same time, the
total NSC in wood collected at the trunk base increased reaching a seasonal peak in September (Figure 3a). This suggests
that these NSC were an important C sink during this seasonal
period. Hence, the present data indicate that the seasonal net
C sink of this beech forest was mostly distributed between
plant growth (May–July) and NSC accumulation in woody parenchymatic cells (August–September). These results are in
agreement with previous observations on beech showing an
increase of wood NSC between bud break and leaf shedding
(Barbaroux and Bréda 2002, Barbaroux et al. 2003) and a
seasonal peak of starch accumulation in stems at the end of
September (Damesin and Lelarge 2003). Coarse roots accumulated significantly higher amounts of NSC compared with
trunks (Figure 5a), indicating that roots play a relevant role as
C sink and reserve pool in this species, consistent with
Barbaroux et al. (2003).
Sampling in 2 years characterized by different climatic conditions (2001 and 2002, Figures 1 and 2) allowed the evaluation of the effects of environmental factors on growth and
carbohydrate accumulation in wood. It is interesting to observe
that despite the relevant climatic differences, trunk radial
growth and NSC accumulation in woody tissues showed very
similar trends in 2001 and 2002 (Figure 3a). However,
whereas trunk growth did not show any relevant differences,
the accumulation of NSC showed some interesting inter-annual
changes (Figure 3a). In particular, our results seem to indicate
that the reduced water availability in July–August 2001 caused
an initial stress associated with a delay in the accumulation of
NSC in sink organs compared with 2002. Indeed, while in
2002 the accumulation of wood NSC started from the end of
July, in 2001 this process was evident only starting from
August (Figure 3a). Consistent with this hypothesis, the predawn leaf water potential in August 2001 was about −1.0 and
−0.8 MPa for top and bottom leaves, respectively, compared
with values of −0.5 and −0.6 MPa for the same month in 2002
(data not shown). Moreover, during the summer period
between July and August, the concentration of leaf soluble sugars at canopy level was lower in 2001 than 2002 (Figure 6b),
suggesting a reduced photosynthetic carbohydrate availability.
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738 Scartazza et al.
However, from August to September 2001, beech plants
showed a higher accumulation rate of reserves than 2002,
although during this seasonal period significantly lower values
of SWC were found (Figure 2a). As a consequence, it is noteworthy that the concentration of reserve carbohydrates in
woody tissues at the end of the growing season was not statistically different between 2001 and 2002 (Figure 3a). Similarly,
Barbaroux and Bréda (2002) found that when water content
was depleted, stem growth of beech plants ceased, whereas
the accumulation of NSC was negligibly affected and continued
until leaf fall. Other authors (Lacointe et al. 2004, Silpi et al.
2007) suggested that, when carbohydrate availability is
reduced, trees tend to adjust the amount of carbohydrate
reserves stored to the level of metabolic demand at the possible expense of growth. Würth et al. (2005) found a higher concentration of NSC in a semi-deciduous tropical forest in the dry
season than in the wet season, while Galvez et al. (2011)
reported an increase of root carbohydrate reserves in droughtstressed aspen seedlings relative to controls. In two Nothofagus
species, drought resistance has been associated with the ability to accumulate reserves (Piper 2011), with a depletion of C
storage in the more drought susceptible species and an
increase of total NSC in the relatively drought-resistant ones.
Woodruff and Meinzer (2011) found that NSC concentrations
were positively correlated with tree height and inversely correlated with shoot midday water potential, shoot osmotic
potential at full turgor and shoot extension, suggesting that
water, and not C, limits growth under moderate drought. It has
been proposed that large storage pools in trees serve to maintain hydraulic transport during episodes of severe stress (Sala
et al. 2012) and that C depletion is more probable in genotypes less adapted to drought (Sala et al. 2010). Hence, our
results seem to indicate that beech plants at the Collelongo
forest are well adapted to drought events and are able to prevent C depletion during the dry season. This could be a relevant adaptive trait of European beech of Southern regions that
might be able to better cope with drought events than beech
of Central European origin (Nahm et al. 2006). However, it is
possible that drought during 2001 was not strong enough to
induce C depletion and a consequent reduction of growth and
reserve accumulation in this forest. Moreover, differences in
climatic conditions between 2001 and 2002 (Figures 1 and 2)
could have affected the balance between photosynthetic C
uptake and respiration of assimilates. This might help to explain
the lack of significant differences in the amount of NSC accumulated in woody tissues at the end of the growing season.
It has been shown that summer drought and increased temperature can determine a concomitant decrease of gross primary productivity (GPP) and ecosystem respiration in
European ecosystems (Ciais et al. 2005), indicating that ecosystem respiration decreases together with GPP, rather than
accelerating with the temperature rise. The decrease of eco-
Tree Physiology Volume 33, 2013
system respiration can be due to a reduction of both microbial and plant respiration with decreasing water availability.
Consistent with this hypothesis, higher values of ecosystem
respiration were recorded during the second part of the season in the wetter year compared with the drier one (200 g
C m−2 in August–September 2002 compared with 100 g
C m−2 in 2001), leading to a decrease of net ecosystem
exchange (NEE) but not of GPP. Moreover, during the 2002
growing season, increased cloudiness resulted in lower values
of incident global solar radiation than 2001 (Figure 2b). In this
respect, previous works on this forest have shown that canopy
light-use efficiency under prevalent conditions of diffuse radiation was higher than under direct solar radiation, although
the net C uptake increased with increasing incident solar radiation during all the seasonal periods (Matteucci 1998). A
direct relationship between GPP and the solar radiation was
observed during 2001 (data not shown), suggesting that the
higher irradiance in 2001 than 2002 could partly counterbalance the negative effect on GPP of the reduced SWC.
Conversely, in 2002, the decreased incident radiation did not
result in lower GPP, probably due to the better hydric status
and the higher proportion of the diffuse radiation component
under which the canopy was more efficient (Matteucci 1998).
As a matter of fact, GPP was not significantly different in the 2
years (1012 g C m−2 in May–September 2002 compared with
1069 g C m−2 in 2001). Hence, these results suggest that C
sink capacity of this forest may depend on a delicate balance
among the effects of several contrasting environmental factors on the individual C fluxes.
Quantitative and isotopic analysis on NSC of trunk
and root woody tissues
The highest seasonal values of soluble sugar content at bud
break (end of April) and after leaf shedding (November) of
both years, associated with the lowest values of starch content
(Figure 3b), indicates an inter-conversion between starch and
wood soluble sugars. Indeed, it is well known that at the onset
of the growing season, beech plants utilize part of the reserve
compounds to sustain leaf growth when these organs are still
not completely autotrophic and current demand exceeds
assimilate supply (e.g., Barbaroux et al. 2003, Damesin and
Lelarge 2003, Silpi et al. 2007). This partly explains the low
starch content recorded at bud break and during the early
spring growth, associated with the high level of soluble sugars
(Figure 3b). The decrease of soluble sugars and the simultaneous increase of starch from the end of April to June suggest an
active starch synthesis in woody tissues (Figure 3a). This result
indicates that growth and C storage were not disconnected, as
previously shown in beech plants (Barbaroux and Bréda 2002,
Damesin and Lelarge 2003). The simultaneous increase of
both starch and soluble sugars in wood from August to
September caused a significant increment of the total wood
Growth, non-structural carbohydrates and δ13C dynamics in beech 739
carbohydrates in both years (Figures 3a and 5a), possibly due
to a high contribution of the recent photosynthetic products to
the reserve accumulation in September. This hypothesis is supported by the high increase of sucrose concentration observed
in wood during this seasonal period (Figure 3b). After leaf
shedding, a decrease of starch associated with a significant
increase of hexoses (glucose and fructose) was recorded, indicating the hydrolysis of starch and sucrose reserves during the
autumn–winter period regulated by temperature (e.g., Sauter
1988). These soluble carbohydrates are important for plant
survival during the leafless winter season, representing the
main C source for cold acclimation, development and
maintenance of cold tolerance and cellular respiration (e.g.,
­
Sauter 1988, Ögren 2000) and may have a role as osmoregulators for freeze protection in drought-stressed plants (Wong
et al. 2009).
The values of δ13C of NSC extracted from woody tissues are
consistent with previous observations (e.g., Damesin and
Lelarge 2003, Göttlicher et al. 2006). Wood carbohydrates
were strongly 13C enriched compared with leaf sugars throughout the season (compare Figures 4 and 5b and Figure 6a),
consistent with other studies (e.g., Brugnoli et al. 1988,
Gleixner et al. 1993, Göttlicher et al. 2006). It is well known
that roots and woody stems are generally isotopically heavier
than leaves due to several possible fractionation processes (for
reviews see Badeck et al. 2005, Cernusak et al. 2009, Werner
and Gessler 2011). Moreover, different values of δ13C between
carbohydrates belonging to source and sink organs have been
previously observed in several plants (for a review see Brugnoli
and Farquhar 2000). For example, sucrose extracted from sink
organs (as root beet) generally shows lower isotopic discrimination than leaf sucrose (Gleixner et al. 1993). This difference
can be attributed to the isotopic effects occurring during postphotosynthetic processes, as the equilibrium isotopic effect
associated with the transport of the trioso-phosphate from the
chloroplast to the cytosol (Brugnoli et al. 1988), the isotopic
effect associated with the reactions catalyzed by aldolase
(Gleixner et al. 1993, Gleixner and Schmidt 1997) and possible isotopic fractionations occurring along the phloem transport path, due to repeated unloading of sucrose, metabolic
conversion of part of this sugar pool and reloading of the
remaining unreacted sugars into the sieve elements (Cernusak
et al. 2009, Brüggemann et al. 2011). The bark sugars, collected after leaf shedding, showed an isotopic signature similar
to that of wood starch (Figures 4 and 5b), suggesting that
these carbohydrate pools are metabolically connected each
other. This is supported by the partial starch degradation that
we observed in wood during the cold season (see Figure 3b).
Our data showed wide seasonal and inter-annual variability
in δ13C in wood soluble sugars, whereas starch showed only
little isotopic changes (Figures 4 and 5b) notwithstanding the
seasonal variation of its concentration (Figure 3b). These
results are in agreement with Damesin and Lelarge (2003) and
suggest a faster turnover rate of soluble sugars, while starch
probably represents a quite stable and isotopically homogeneous carbon pool. The seasonal and inter-annual variability in
δ13C of wood soluble sugars can be attributed to several
causes related to photosynthetic and post-photosynthetic processes: (i) possible post-photosynthetic fractionation processes related to starch synthesis and remobilization in woody
tissues; (ii) possible fractionation processes during respiration
of root and trunk woody tissues; and (iii) seasonal and interannual variations of climatic conditions. Consistent with the
first hypothesis, a 13C enrichment of wood soluble sugars was
observed from bud break to June in both years, when a depletion of soluble sugars associated with starch synthesis
occurred in woody tissues; conversely, a depletion in 13C in
wood soluble sugars was observed in the late part of the vegetative season and after leaf shedding, when starch was hydrolyzed and soluble sugars concentration sharply increased.
During these seasonal periods the composition of the whole
soluble sugars changed, with an increase of the ratio between
sucrose and (glucose + fructose) during the starch synthesis
and a decrease of this ratio during the starch hydrolysis (Figure
3b). Damesin and Lelarge (2003) showed that these soluble
carbohydrates were characterized by different isotopic signature; hence, it is possible that a different contribution of
sucrose, fructose and glucose to the overall soluble sugars
pool during different seasonal periods and/or fractionation processes during the inter-conversion among these compounds
can modify the isotopic signature of total wood soluble carbohydrates. Moreover, we cannot exclude a possible fractionation
during the transport of hexoses inside the plastids for starch
synthesis. For example, Feng and Tang (2011) suggested that
glucose transporters in cell membranes of Escherichia coli may
favor light glucose molecules, causing isotopic fractionation
during glucose uptake. Hence, a fractionation favoring the light
C isotope during the transport of glucose through the plastid
envelope membrane for starch synthesis could modify the isotopic composition of the remaining sugar pools. Fractionation
processes occurring during respiration of wood soluble sugars
might also contribute to the observed isotopic variations. It has
been demonstrated that heterotrophic tissues, opposite to the
leaves, generally release 13C-depleted CO2, thereby leaving
behind 13C-enriched carbon (Badeck et al. 2005, Cernusak
et al. 2009, Werner and Gessler 2011). This is consistent with
the fact that soluble sugars represent one of the most important respiratory substrates in roots and trunks; hence respiration of sugars might release 13C-depleted CO2 leaving behind
13C-enriched sugars in woody tissues. Moreover, possible seasonal changes of respiratory substrates might also modify δ13C
of ecosystem respired CO2 and of the remaining reserve C
pools (Damesin and Lelarge 2003). Finally, variation of δ13C in
wood soluble sugars is partly associated with seasonal
Tree Physiology Online at http://www.treephys.oxfordjournals.org
740 Scartazza et al.
changes of rainfall, temperature and SWC (Figures 1 and 2a).
In particular, higher rainfall and SWC in 2002 than 2001 probably caused an increase of canopy Δ and hence a decrease in
δ13C of photosynthetic products transported towards sink
organs during the August–September period. Consistent with
this hypothesis, the inter-annual isotopic difference for wood
soluble sugars in September (~1.6‰) was very similar to that
observed for canopy leaf soluble sugars (compare Figures 4
and 6a). Then, although it is probable that a combination of the
above-mentioned mechanisms affects δ13C values of wood
soluble sugars, it is still possible to distinguish the effect of a
dry seasonal period on their isotopic signature.
Quantitative and isotopic analysis on canopy leaf
soluble sugars
Based on C discrimination model (Farquhar et al. 1982,
Brugnoli et al. 1988, Brugnoli and Farquhar 2000), higher δ13C
values in both leaf and wood soluble sugars during August–
September 2001 compared with 2002, indicate a parallel
decrease in the ratio between intercellular and atmospheric
partial pressure of CO2 (pi/pa) in canopy leaves. This interannual difference of isotopic signature in NSC may be explained
by partial stomatal closure induced by decreasing SWC during
the dry periods. Consistent with this hypothesis, a highly significant linear relationship was found between δ13Ccanopy and
SWC for both the years (Figure 7). Hence, the reduced SWC
during the second part of the 2001 growing season caused a
progressive enrichment in 13C of both leaf and wood soluble
sugars, indicating a decrease of canopy photosynthetic Δ and,
indirectly, an increase of WUE (Farquhar et al. 1989, Brugnoli
and Farquhar 2000). Conversely, during August–September
2002, soil water availability was not a limiting factor, possibly
leading to high values of stomatal conductance and, consequently, to high canopy Δ and low WUE. Hence, our isotopic
data highlight a significantly higher WUE in 2001 than 2002,
particularly during the second part of the vegetative season
when plants accumulated large amounts of carbohydrate
reserves in storage organs. Enhanced WUE, consequent to
partial stomatal closure, is a common response of plants
exposed to drought. This may be a relevant trait for maintaining
an acceptable plant water status and a relatively high C sink
capacity in this beech forest, as indicated by correspondingly
high values of NEE (Scartazza et al. 2004) and carbohydrate
reserve accumulation in sink organs (Figure 3b). The reduced
SWC during the dry year caused an initial decrease in the canopy-integrated value of leaf soluble sugar concentration from
June to August (Figure 6b). However, it is noteworthy that the
amount of canopy soluble sugars in September showed only
slight non-significant variations, although marked differences in
SWC and δ13Ccanopy between 2001 and 2002 were evident.
Hence, these results suggest that this forest was able to maintain similar amounts of carbohydrates in both source and sink
Tree Physiology Volume 33, 2013
organs between 2001 and 2002 during the C storage period,
further supporting the hypothesis that in the relatively dry
period it was able to prevent C depletion.
Conclusion
Our results indicate that storage carbohydrates represented a
consistent C sink during the late summer–autumn period.
Quantitative and isotopic analysis of NSC indicated that the
studied forest responded to variability in environmental factors
during different growing seasons, with short to medium term
homeostatic equilibrium. This allowed it to maintain similar
growth and NSC dynamics during years with different water
availability and solar radiation. These results suggest the presence of efficient internal regulation mechanisms in Southernadapted beech plants, ensuring a favorable physiological status
under the relatively dry Mediterranean climate. Our results
highlight the role of reserves for plant survival and for resilience processes in drought-prone environments. This information may be useful for beech forest management in the
Mediterranean area under the expected future climatic
changes. In this respect, it will be crucial to study in the longterm how the increasing frequency and intensity of drought
events can affect productivity and functionality of beech forests and their C sequestration potential in the Mediterranean
Region.
Acknowledgements
The authors thank Luciano Spaccino for the skilful assistance
in plant and air collection and mass spectrometer analysis. The
Collelongo site is part of the Italian network for Long Term
Ecological Research sites (LTER-Italy).
Funding
This work was supported in part by the European Community’s
Potential Programme Netcarb (HPRN-CT-1999-00059), by the
European Union projets ForCast (EVK2-1999-00242) and
CarboEuroflux (EVK2-1999-00229) and by the Italian Forest
Monitoring Programme ‘Conecofor’ coordinated by the National
Forest Service of Italy (CFS).
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