RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2006; 20: 219–226
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2297
13
C/12C isotope labelling to study leaf carbon respiration
and allocation in twigs of field-grown beech trees
Salvador Nogués1*,{, Claire Damesin1, Guillaume Tcherkez2,
Florence Maunoury1, Gabriel Cornic1 and Jaleh Ghashghaie1
1
Laboratoire d’Ecologie Systématique et Evolution, Unité Mixte de Recherche 8079, Université de Paris XI, Bâtiment 362, 91405 Orsay, France
Laboratoire Structure et Métabolisme des Plantes, Institut de Biotechnologie des Plantes, Université Paris XI, Bâtiment 630, 91405 Orsay,
France
2
Received 24 September 2005; Revised 13 November 2005; Accepted 13 November 2005
In situ 13C/12C isotopic labelling was conducted in field-grown beech (Fagus sylvatica) twigs
to study carbon respiration and allocation. This was achieved with a portable gas-exchange open
system coupled to an external chamber. This method allowed us to subject leafy twigs to CO2
with a constant carbon isotope composition (d13C of 51.2%) in an open system in the field.
The labelling was done during the whole light period at two different dates (in June 2002
and October 2003). The d13C values of respiratory metabolites and CO2 that is subsequently
respired during the night were measured. It was found that night-respired CO2 is not completely
labelled (only ca. 58% and 27% of new carbon is found in respired CO2 immediately after the labelling in June 2002 and October 2003, respectively) and the labelling level progressively disappeared
during the next day. It is concluded that the carbon respired by beech leaves after illumination was
supplied by a mixture of carbon sources in which current carbohydrates were not the only contributors. In addition, as has been found in herbaceous plants, isotopic data before labelling showed
that carbon isotope discrimination favoring the 13C isotope occurred during the night respiration of
beech leaves. Copyright # 2005 John Wiley & Sons, Ltd.
Determining the carbon balance of the terrestrial ecosystem
and understanding how it depends on the environmental
conditions are currently major objectives of the scientific
community (e.g. AMERIFLUX and CARBOEUROFLUX projects). In this context, forests are particularly important as
they represent 80% of the terrestrial biomass.1 As a consequence, currently considerable effort has been made to
understand forest growth and pathways of carbon fluxes in
forest ecosystems. Carbon input in the system via tree photosynthesis has been well characterized and simulated using
the biochemical Farquhar’s model on leaf assimilation.2 The
carbon (C) output (i.e. respiration) has also been characterized. The processes have been described at the ecosystem
level and also on an annual scale.3– 5 In contrast, allocation
of assimilated C (i.e. its distribution in the different tree tissues) has received less attention. The process has thus been
studied in simulations. Knowledge of the distribution of C
in individual trees is considered essential to a proper understanding of forest growth.
*Correspondence to: S. Nogués, Departament de Biologia Vegetal,
Universitat de Barcelona, 645 Diagonal Av, 08028 Barcelona,
Spain.
E-mail: [email protected]
{
Present address: Departament de Biologia Vegetal, Universitat
de Barcelona, 645 Diagonal Av, 08028 Barcelona, Spain.
Contract/grant sponsor: European Community’s Human Potential Program; contract/grant number: HPRN-CT-1999-00059,
NETCARB.
Labelling techniques using 14C or 13C have been used for
many years to study the allocation of carbohydrates
produced by photosynthesis between different parts of the
plant, especially on herbaceous species grown in controlledenvironmental conditions6–11 or in natural abundance conditions.12 Lacointe and coworkers13 have tested the branch
autonomy theory inside tree crowns (i.e. the direct effect of
light on photosynthesis is not compensated for by carbohydrate exchanges between branches of different light status)
using a 13C/14C double-labelling experiment on 3-year-old
walnut trees. They found that in September (in the northern
hemisphere) branch autonomy was nearly total (i.e. there was
no exchange between branches); however, in winter, significant amounts of C were imported by branches (representing up to 10% of total branch reserves). Nevertheless, data on
the precise partitioning of photo-assimilated C between
distinct parts on adult trees in the field are very scarce in the
literature.
Direct measurements of the 14C or 13C labelling in respired
CO2 in trees are surprisingly limited,14,15 and, as far as we
know, such measurements have never been carried out on
adult trees in the field. It has been recently shown on
herbaceous species grown in controlled conditions that
most of the C released by dark respiration in leaves after
a period of light does not come from C recently assimilated
by photosynthesis.11 Because of the complexity of tree
structure and of possible interactions between autotrophic
(mainly leaves) and heterotrophic (i.e. trunk, roots, etc.)
Copyright # 2005 John Wiley & Sons, Ltd.
220
S. Nogués et al.
tissues, it is possible that such a result is not valid in
ligneous plants. In particular, C stored in branches and
trunk (as reserves) may act as a sink and a source of C
depending on the season and growth conditions, thus
blurring the picture seen in herbaceous plants. For example,
Barbaroux and Bréda16 showed on beech trees that accumulation of some reserves occurred simultaneously with growth
during the vegetative period. Damesin and Lelarge17 showed
that a portion of stored C (naturally enriched in 13C) is used in
the initial phase of leaf and stem construction and during
winter respiration.
In this study, a 13C/12C isotope labelling technique
was used to study the allocation of recently assimilated C by
photosynthesis at short term (over 2 days) and further night
respiration after the photoperiod in adult trees of
Fagus sylvatica. An experimental set-up using a portable
gas-exchange open system LI-6400 (LICOR Inc., Lincoln, NB,
USA) was adapted for in situ carbon labelling. We have
taken advantage of the difference in the C isotope composition (d13C) between atmospheric CO2 (10.5%) and
commercially available 12C-enriched CO2 (51.2%), and
consequently the 13C abundance in the CO2 used for
the labelling is of the same order of magnitude as that
found in nature. This allows one to pick up the contribution
of stored C vs. current photosynthates to CO2 production
by respiration. It is noteworthy that this would not have
been possible if heavily labelled C had been used (i.e. several
percent in 13C would have blurred the contribution of
non-labelled C). The objective of the study was to examine
the contribution of leaf-assimilated C to respiratory losses
(i.e. CO2), leaf metabolite (soluble sugars and starch)
production and carbohydrate export from stems. The
labelling was achieved on current-year twigs at two
contrasted periods of the tree life (at the end of leaf growth
in June and at the end of leaf lifespan in October).
EXPERIMENTAL
Plant material and experimental site
Three (isolated) 20-year-old beech (F. sylvatica) trees grown in
the campus of the University of Paris XI at Orsay, France
(488420 N, 028100 E) were used in this study. A complete
description of the plant material and the experimental site
has been published.17 Mean annual precipitation, and minimum and maximum temperatures of the site are 685 mm,
78C and 168C. Photosynthetically active photon flux density
(PPFD) and air temperature (Tair) were measured during the
two dates: 18–19 June 2002 and 17–18 October 2003 (Fig. 1).
The d13C of the air CO2 surrounding the trees was
10.5 0.4% (average of the two measuring periods). Sunexposed leaves in twigs and current-year produced stems
(Sn, i.e. S2002 or S2003, respectively) at the bottom of the
crown were used for the isotopic labelling. The labelling
was also followed in the stem (of the same branch) grown
during previous years.
Gas-exchange and C-labelling procedures
For both measuring periods, the same sequence was monitored on one (attached) twig of the bottom of the crown
(including 6 sun-exposed leaves and the current year stem):
(i) sampling of respired CO2 at predawn; (ii) labelling during
the light time of the day (i.e., ca. 12 and 8 h labelling periods
for June 2002 and October 2003, respectively); and (iii)
sampling of respired CO2 immediately after the labelling
and three following times the day after (i.e. at the end of
the first night, in the middle of the second day, and at the
end of the second day). At the end of the experiment (i.e.
beginning of the second night), the labelled leaves and
the corresponding current-year stem, the 1-year-old and
2-year-old stems and the nearest current year ramification
present on the 1-year-old stem (see Figs. 4 and 5 for schemes)
35
2000
June 2002
1800
30
25
1400
1200
o
20
Tair ( C)
PPFD ( mol m-2 s-1)
1600
1000
15
800
600
10
October
2003
400
5
200
0
0
0
10
20
30
40
Time (h)
Figure 1. Photosynthetically active photon flux density (PPFD, circles) and air temperature
(Tair , squares) during the two measuring periods (18–19 June 2002, closed symbols, and 17–
18 October 2003, open symbols) in the campus of the University of Paris Sud at Orsay, France.
Copyright # 2005 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2006; 20: 219–226
Carbon isotope labelling in trees
were sampled for isotopic analysis. Non-labelled twigs of the
same trees were also sampled as control.
Closed system for dark respiration
Before the labelling, attached leaves (typically six) of a twig
were placed in a respiration chamber for measurements of
dark-respired CO2 (unlabelled). The respiration chamber
was first ventilated with CO2-free air and then the CO2
respired was allowed to accumulate in the chamber.18 The
gas contained by this chamber was introduced into a 50-mL
syringe (Magnum syringe; Alltech France, Carquefou,
France). The sample gas in the syringe was then introduced
into the 15-mL gas sample loop of an elemental analyser
(EA, NA-1500; Carlo-Erba Instruments, Milan, Italy),19 and
the isotope analysis was conducted in the continuous flow
mode. Briefly, the gas contained by the loop was introduced
into the EA with helium for gas chromatography through a
six-way valve. The connection valve between the EA and
the isotope ratio mass spectrometer (IRMS, VG Optima,
Micromass, Villeurbanne, France) was opened just for the
period of time that allows CO2 isotope analysis. Carbon isotope compositions were calculated as deviations of the C isotope ratio (13C/12C, called R) from the international standard
(Pee Dee Belemnite):
13 Cð%Þ ¼ 103 ½ðRsample Rstandard Þ=Rstandard :
Open system for isotopic labelling
After the initial measurement of dark-respired CO2, the leafy
twig was removed from the respiration chamber and placed
(still attached) in a gas-exchange chamber as previously
described.11 The stem in the chamber was already covered
(during the initial measurement of dark-respired CO2) by
aluminum foil to avoid stem photosynthesis (stem photosynthesis in beech has been shown to occur20). The chamber
was connected to the sample air hose of the LI-6400 portable
gas-exchange system just before the (usual) leaf-clip chamber. The latter is empty and closed. The gas-exchange chamber is made of aluminum (20 cm 12 cm 6 cm), with a clear
plastic lid, and accommodates the six leaves of the twig (total
leaf surface ca. 0.01 m2). Two fans were enclosed in the chamber and gave a boundary layer conductance to water of
ca. 6.7 mol m2 s1. The temperature of the chamber was controlled at ca. 208C with circulating water from a cooling water
bath to the jacket of the leaf chamber, and the leaf temperature
was measured with a copper-constantan thermocouple
plugged to the thermocouple sensor connector of the LI6400 leaf-clip chamber. The ingoing air flow was 1 L min1
and was monitored by the LI-6400. The molar fractions of
CO2, the humidity and the light were measured with the
infrared gas analyser (IRGA) and the light sensor of the
LI-6400. The CO2 used for labelling was obtained from a
bottle (Air Liquide, Grigny, France) with a d13C value of
51.2 0.1%. The d13C value of the ingoing air was also
regularly checked by taking a sample into a glass balloon
(see below).
When the photosynthetic measurements were made, the
outgoing air of the chamber was no longer directed to the
IRGA but instead to 150-mL glass balloons (Scott Glass,
Mainz, Germany). Balloons were taken to the lab, and the
Copyright # 2005 John Wiley & Sons, Ltd.
221
sample gas was introduced into the sample loop of the
EA using a pump. The on-line photosynthetic discrimination (Dobs) was calculated using the following
relationship:21
obs ¼ ðe o Þ=½1 o þ ðe o Þ;
where do and de are the d13C values of outlet and inlet air, and
x ¼ ce/(ce co), where ce and co are the inlet and outlet CO2
molar fractions.
After the labelling, leaves were removed from the
gas-exchange chamber and were placed back into the
respiration chamber for measurements of dark-respired
CO2 (labelled) as described above. At the end of the
measurements (i.e. at the beginning of the second night),
leaves and stems were immediately frozen in liquid nitrogen.
They were then lyophilized, and powdered for metabolite
analysis.
Carbohydrate extractions and quantification
The starch and sucrose, glucose and fructose extraction procedures were as previously described.17,19 Briefly, leaf and
stem powder was suspended with 1 mL of distilled water
in an Eppendorf tube (Eppendorf Scientific, Hamburg,
Germany). After centrifugation, starch was extracted from
the pellet by HCl solubilization. Soluble proteins of the
supernatant were heat denatured and precipitated and
soluble sugars and organic acids of the protein-less extract
were separated by high-performance liquid chromatography
(HPLC). After lyophylization, purified metabolites were
suspended in distilled water, transferred to tin capsules
(Courtage Analyze Service, Mont Saint-Aignan, France) and
dried for isotope analysis. Isotope analysis of metabolites was
conducted using the same EA and IRMS as were used for gas
measurements.
RESULTS
The environmental conditions during the two measuring
dates (i.e. 18–19 June 2002 and 17–18 October 2003) were
typical of the temperate climate under oceanic influence of
the Paris region (Fig. 1). June 2002 was warm (maximum
PPFD and Tair values were 1900 mmol m2 s1 and 328C,
respectively) and October 2003 was relatively cold (maximum PPFD and Tair values were 1200 mmol m2 s1 and
118C, respectively).
Labelling in dark-respired CO2
The d13C of respired CO2 in the leafy twig of beech trees was
ca. 18.5% before the labelling (measured in the closed system) at the end of the night. Then the leafy twig was placed in
the open system for isotopic labelling with CO2 depleted in
13
C (d13C of 51.2%) during a sunny day in June 2002
(Fig. 2) or October 2003 (Fig. 3). During the experiment conducted in June 2002, beech leaves assimilated ca. 300 mmol C
per m2 during the day (A was ca. 6.5 mmol m2 s1, Fig. 2).
Low light intensities produced lower assimilation rates during October 2003 (Fig. 3) than during June 2002 (Fig. 2). It
should be also noted that, although Tair was different during
the two measuring periods and variable during the day, leaf
temperature was maintained at 208C in order to maintain
Rapid Commun. Mass Spectrom. 2006; 20: 219–226
S. Nogués et al.
-51.2‰
-10.5‰
A
10
C (‰)
6
13
15
8
4
5
2
0
RD ( mol m-2 s-1)
20
10
0
B
-0,5
-10
-1,0
-20
-1,5
-30
-2,0
-40
-2,5
-50
5
10
15
20
25
30
35
40
45
C of resp. CO2 (‰)
A ( mol m-2 s-1)
12
13
222
50
Time (h)
Figure 2. Changes in the photosynthetic rate (*, mmol m2 s1), D13C (*, %), dark-respiration
rates (&, mmol m2 s1) and d13C of respired CO2 (&, %) in leafy twigs of beech trees during
June 2002 for two consecutive days. The top bar indicates the night and day periods, respectively
(during the day the d13C of CO2 given to the leaves is also shown). Data are the means of two
replicates (standard deviations are shown when larger than the symbols).
-51.2‰
-10.5‰
A
4
10
2
5
0
0
RD ( mol m-2 s-1)
B
-10
-0,5
-20
-1,0
-30
-40
-1,5
5
10
15
20
25
30
35
40
45
13
15
C of resp. CO2 (‰)
6
C (‰)
20
13
A ( mol m-2 s-1)
8
50
Time (h)
Figure 3. Changes in the photosynthetic rate (*, mmol m2 s1), D13C (*, %), darkrespiration rates (&, mmol m2 s1) and d13C of respired CO2 (&, %) in leafy twigs of beech
trees during October 2003 for two consecutive days. The top bar indicates the night and day
periods, respectively (during the day the d13C of CO2 given to the leaves is also shown). Data
are the means of two replicates (standard deviations are shown when larger than the symbols).
similar stomatal and photo-respiratory conditions on the
leaves during the labelling.
Leaf respiration increased after sunset (to ca. 1.5 mmol
m2 s1) and progressively decreased during the night
(to ca. 0.5 mmol m2 s1). During the whole night of June
2002, leaves respired ca. 100 mmol C per m2; this is one-third
Copyright # 2005 John Wiley & Sons, Ltd.
of the C assimilated during the day. This dark-respired CO2
was labelled, i.e., d13C of the evolved CO2 was 44.5% at the
beginning of the night and 48.3% at the end of the night
(Fig. 2). During June 2002, the (photosynthetic) C isotope
discrimination (D13C) was ca. 15% in the morning as stomata
were still opening (this corresponds to a pi/pa value ca. 0.5,
Rapid Commun. Mass Spectrom. 2006; 20: 219–226
Carbon isotope labelling in trees
data not shown) and reaches 20% during the day (i.e. pi/pa
value ca. 0.7). As the average D13C of the leaves was ca. 17%,
and the d13C of dark-respired CO2 by leaves may have been
13
C-enriched by edark 4–6%22 (compared with the respiratory substrates), the d13C of the respiratory substrates (mainly
carbohydrates) should have approached 64% (i.e.
51 D13C þ edark) if the substrates used to sustain respiration had been fully labelled (with a CO2 of d13C of 51%), but
this was not the case (Fig. 2). Furthermore, as expected, the
d13C value of respired CO2 progressively increased to
27.2% at the beginning of the second night; during the
second day, leaves assimilated a similar amount of nonlabelled CO2, i.e. with a d13C value of 10.5%.
Shorter light periods and a lower light intensity in October
2003 led to a lower A of ca. 3.5 mmol m2 s1 (Fig. 3) of the leafy
twig. During a sunny day in October 2003, leaves assimilated
ca. 100 mmol C per m2. During the whole night, leaves
respired ca. 50 mmol C per m2; this is half of the C assimilated
during the day. Dark-respired CO2 was less labelled (i.e.
32.3% at the beginning of the night and 24.8% at the end
of the night, Fig. 3) than in June 2002. The labelling in the
respired CO2 progressively decreased, with a d13C value of
23.7% at the beginning of the second night (during the
second day leaves assimilated a similar amount of nonlabelled C, i.e. at a d13C of 10.5%).
Labelling in metabolites
It is noteworthy that the leaves are sampled at the end of the
measurement period (i.e. at the beginning of the second
223
night) and so the isotope composition of the organics reflects
both the labelling (with a d13C value of 51.2% during the
first day) and its dilution by subsequent assimilation (during
the second day leaves assimilated a similar amount of nonlabelled CO2, i.e. with a d13C value of 10.5%).
At the end of the measurement period, all the metabolites
(sucrose, glucose, fructose and starch) of the leaves in the
labelling chamber were still labelled both in June 2002 (they
are ca. 5% 13C-depleted) and October 2003 (ca. 4% 13Cdepleted, Figs. 4 and 5) compared with the non-labelled
(control) leafy twigs (control values are in the figure legends).
However, metabolites are less labelled in October than in
June, simply because of a lower assimilation and smaller
photosynthetic isotope discrimination (Figs. 2 and 3). By
contrast, the nearest (lateral) current-year ramification (Sn) in
Figs. 4 and 5 are not labelled, having isotope abundances
similar to the control. We recognize that several parameters
such as their temperature were not controlled and, consequently, the photo-respiratory flux was not also. This may
induce a different photosynthetic discrimination that could
compensate for the transfer of labelled C from the labelled
branch. However, metabolites of the lateral current-year
ramification are not depleted in 13C after the labelling and we
assume here that it reflects no labelling, i.e. no transfer of 13Cdepleted products from the labelled branch.
The leaf total organic matter (TOM) was slightly labelled
(by ca. 2%) in the leaves (in the chamber, i.e. 2002). Further,
metabolites were also labelled in the current-year stem in
spring (Sn, i.e. S2002, Fig. 4), but this is not the case for the
Figure 4. Schematic representation of the d13C of sucrose, glucose, fructose, starch and
TOM (from left to right) of leaves and stems during June 2002 at the end of the experiment
(i.e. beginning of the second night). We labelled the leaves of the S2002 (Sn) supplied with
an air d13C of 51.2% and we followed the labelling in the S2001 (Sn-1) and S2000 (Sn-2)
and in leaves and stems of the neighboring stem S2002 (Sn). ‘n.d.’ means that the d13C
was not determined due to lack of enough purified metabolites. Amounts of the different
metabolites are also indicated in brackets [% of dry weight]. Control (non-labelled) values
for sucrose, glucose, fructose, starch and TOM were 24.7, 24.9, 25.0, 24.8, and
27.8% for the leaves and 23.1, 22.6, 24.4, 23.5, and 26.2% for the stem
(S2002), respectively. Data are the means of two replicates (standard deviations are lower
than 10% in all cases).
Copyright # 2005 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2006; 20: 219–226
224
S. Nogués et al.
Figure 5. Schematic representation of the d13C of sucrose, glucose, fructose, starch and
TOM (from left to right) of leaves and stems during October 2003 at the end of the experiment
(i.e. beginning of the second night). We labelled the top leaves of the S2003 (Sn) with an air
d13C of 51.2% and we followed the labelling in the S2002 (Sn-1) and S2001 (Sn-2) and in
leaves and the stem of the neighboring stem S2003 (Sn). ‘n.d.’ means that the d13C was not
determined due to lack of enough purified metabolites. Amounts of the different metabolites
are also indicated in brackets [% of dry weight]. Control (non-labelled) values for sucrose,
glucose, fructose, starch and TOM were 25.4, 25.6, 26.4, 24.9, and 28.1% for the
leaves and 23.9, 24.5, 25.1, 23.0, and 25.7% for the stem (S2003), respectively.
Data are the means of two replicates (standard deviations are lower than 10% in all cases).
current-year stem 2003 (Sn) in autumn (Fig. 5). Metabolites in
Sn-1 (i.e. S2001, Fig. 4) were slightly labelled. It is interesting
to note that sucrose and starch are the most abundant
carbohydrates in leaves (ca. 7.1% and 4.7% of TOM,
respectively) during June 2002 (Fig. 4). Leaves had their
maximum sucrose values in summer when their glucose and
fructose levels were the lowest.17 Fructose and starch are the
most abundant carbohydrates in the stem (Fig. 4). During
October 2003, starch decreases in the leaves and further
increases in the stem (Fig. 5).
DISCUSSION
The relationships between leaf photosynthesis
and respiration in trees
After ca. 300 mmol C per m2 (June 2002) or ca. 100 mmol C per
m2 (October 2003) had been assimilated by leaves of fieldgrown beech trees during the day, the night-respired CO2
was labelled, indicating that a pool of respiratory C was fed
by photosynthesis and used by respiration in the dark. This
pool may be carbohydrates in the cytosol, although transitory
starch may be also rapidly mobilized (i.e., after 75 min) in the
dark after a light period.23 Another possibility is the remobilization of sucrose stored in the vacuole contributing to the
respiration feeding.24,25 Further experiments are needed to
specify the contribution of each carbohydrate pool to feed
Copyright # 2005 John Wiley & Sons, Ltd.
the respiratory pool. Nevertheless, it can be noted that the
d13C value of CO2 produced the second day is closer to the
d13C value of starch (in both June and October) than it is to
the Suc or Glc values, suggesting that starch might be the
main contributor to respiration feeding in darkness.
All the C used by leaf respiration does not come from recent
assimilates. It is interesting to note that there was only ca. 58%
of new C in respired CO2 in June 2002. This value decreases to
27% in October 2003 (for calculations of the percent of new
carbon in respired CO2, see Nogués et al.11). This implies that
non-labelled C atoms (e.g. from ‘old’ starch) were still feeding
night respiration after the labelling. This effect has been
shown on the herbaceous species Phaseolus vulgaris grown in
controlled conditions.11,26 As previously suggested,11 night
respiration in leaves is fed by a mixture of ‘new’ and ‘old’
carbon, the contribution of the latter being as high as 50%
after 16 h of illumination. Clearly, this also applies to CO2
production in the dark by beech leaves grown in field
conditions. Interestingly, in June (Fig. 2), the d13C of the C in
the respiratory CO2 was the lowest at the end of the first
night suggesting that a first respiratory pool was filled up
first by some newly photosynthesized C, and that another
respiratory pool was then fed slowly from the first one. In
October (Fig. 3) there was an indication of such a process
during the first hour of the night where there is a decline in
the d13C of the respiratory CO2. The comparatively high d13C
value at the end of the night presumably results from the low
Rapid Commun. Mass Spectrom. 2006; 20: 219–226
Carbon isotope labelling in trees
rate of photosynthesis during the previous day with the
consequence of a low labelling in the first respiratory pool.
Fractionation during dark-respiration
in tree leaves
On one hand, photosynthetic CO2 assimilation discriminates
against 13CO2, so the fixed carbon is ca. 20% depleted in 13C
compared with atmospheric CO2.27 On the other hand, it has
been recently shown on herbaceous greenhouse-grown
plants that discrimination occurs during night respiration
leading to the production of 13C-enriched CO2 (between 4
and 6% compared with sucrose22). Before the labelling, the
d13C of respired CO2 in attached leafy twigs of beech trees
was 13C-enriched (ca. 18.5%, Figs. 2 and 3) compared
with the respiratory metabolites and TOM of the leaves (ca.
24 and 27%, respectively, control values in the legends
of Figs. 4 and 5). Such apparent discrimination during dark
respiration in tree leaves grown in field conditions has been
also recently shown by other workers.28 They demonstrated
that there is considerable short-term variation in the leafrespired CO2 in forest canopy (although leaves were
removed from the trees), and that dial changes in leaf carbohydrate status could be used to predict changes in the leafrespired CO2 empirically. The d13C value of CO2 respired
by twigs was (at least for the periods considered) even higher
than have been measured on current-year stems in the same
species (in Damesin and Lelarge17 leaves were not measured
and maximal stem d13C values were 22.1%). It has also
recently been shown that leaf respiratory CO2 is 13C-enriched
(relative to TOM) in five species of C3 trees grown under controlled environmental conditions.29 So 13C enrichment in
dark-respired CO2 seems to be a general feature in Angiosperms. Tcherkez and coworkers19 proposed that this isotopic
discrimination during respiration can be explained by (i) the
C source used for respiration, (ii) the isotope effects of some
respiratory enzymes, and (iii) the non-statistical C isotope
distribution in glucose. The prevalence of the latter led to
use of the term ‘fragmentation fractionation’ to describe the
discrimination associated with dark-respiration.30
Metabolite synthesis in tree leaves
Variations in carbohydrate contents found in June and
October were in agreement with dynamics previously observed in beech.16,17 In leaves, progressive decrease in starch
content throughout the season may be due to an increase of
sink strength and/or to a decrease of the photosynthetic capacity. So, starch accumulated in the stem after the summer. It is
worth noting that large variations occurred in the starch content, whereas the d13C remained quite stable, suggesting that
the starch C pools were rather homogeneous.17
In June, metabolites in Sn-1 (i.e. S2001) were labelled,
indicating that new C was exported from the leaves of the
current-year stems (S2002) to 1-year-old stems (S2001).
According to Lacointe and coworkers,13 this within-stem C
transfers, occurring in June, abolishing branch autonomy
during this period of the year. Furthermore, in October,
between-branch C movements were very limited. In loblolly
pine, it has been also shown that shoots were usually
autonomous with respect to carbohydrate supply, but that
Copyright # 2005 John Wiley & Sons, Ltd.
225
the carbohydrate movements into the terminal shoot from
subtending foliage could occur under conditions of very high
stress (i.e. high growth demand).31 Unfortunately, only
mature needles, excluding growing organs and mature stem
tissues, were evaluated as possible sinks for between-branch
transfers, so their conclusion should be considered partial.
Furthermore, Yamamoto and coworkers32 showed that a
very small quantity of 13C photoassimilates was exported
into a neighboring lateral branch for 44–72 h after a basal or
terminal lateral branch was exposed to 13CO2 labelling, but,
again, due to individual variability, the results were not very
conclusive.
CONCLUSIONS
The 13C/12C isotopic labelling technique allowed us to subject leafy twigs of beech trees to CO2 with a constant C isotope
composition (i.e. d13C of 51.2%) in an open system in the
field, in order to study leaf carbon respiration and allocation.
The d13C values of respiratory metabolites and CO2 that is
subsequently respired during the night were measured. It is
found that night-respired CO2 is not completely labelled
(only ca. 58% and 27% of new carbon is found in respired
CO2 immediately after the labelling in June 2002 and October
2003, respectively) and the labelling level progressively disappeared during the next day. Consequently, the C respired
after illumination by beech leaves was supplied by a mixture
of C sources in which current carbohydrates were not the
only contributors. Furthermore, isotopic data before labelling
showed that a C isotope discrimination favoring the 13C isotope occurred during night respiration of beech leaves.
Acknowledgements
We are grateful to Marc Berry for setting up the gasexchange/IRMS coupling. We thank Max Hill for technical
assistance with the HPLC procedure. This work was supported by the European Community’s Human Potential
Program under contract HPRN-CT-1999-00059, NETCARB.
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