Tree Physiology 35, 1192–1205
doi:10.1093/treephys/tpv096
Research paper
The relationship between needle sugar carbon isotope
ratios and tree rings of larch in Siberia
K.T. Rinne1,5,6, M. Saurer1, A.V. Kirdyanov2, N.J. Loader3, M.V. Bryukhanova2, R.A. Werner4
and R.T.W. Siegwolf1
1Laboratory
of Atmospheric Chemistry, Paul Scherrer Institute (PSI), CH-5232 Villigen, Switzerland; 2V.N. Sukachev Institute of Forest SB RAS, Akademgorodok, Krasnoyarsk
660036, Russia; 3Department of Geography, Swansea University, Singleton Park, Swansea SA2 8PP, UK; 4Institute of Agricultural Sciences, ETH Zurich, Zurich, Switzerland;
5Present address: Natural Resources Institute Finland, PO Box 18, FI-01301 Vantaa, Finland; 6Corresponding author ([email protected])
Received April 29, 2015; accepted August 17, 2015; published online October 3, 2015; handling Editor Lucas Cernusak
Significant gaps still exist in our knowledge about post-photosynthetic leaf level and downstream metabolic processes and isotopic fractionations. This includes their impact on the isotopic climate signal stored in the carbon isotope composition (δ13C) of
leaf assimilates and tree rings. For the first time, we compared the seasonal δ13C variability of leaf sucrose with intra-annual,
high-resolution δ13C signature of tree rings from larch (Larix gmelinii Rupr.). The trees were growing at two sites in the continuous
permafrost zone of Siberia with different growth conditions. Our results indicate very similar low-frequency intra-seasonal trends
of the sucrose and tree ring δ13C records with little or no indication for the use of ‘old’ photosynthates formed during the previous year(s). The comparison of leaf sucrose δ13C values with that in other leaf sugars and in tree rings elucidates the cause for
the reported 13C-enrichment of sink organs compared with leaves. We observed that while the average δ13C of all needle sugars
was 1.2‰ more negative than δ13C value of wood, the δ13C value of the transport sugar sucrose was on an average 1.0‰ more
positive than that of wood. Our study shows a high potential of the combined use of compound-specific isotope analysis of
sugars (leaf and phloem) with intra-annual tree ring δ13C measurements for deepening our understanding about the mechanisms
controlling the isotope variability in tree rings under different environmental conditions.
Keywords: carbohydrates, Central Siberia, climate, compound-specific, Larix gmelinii (Rupr.), laser ablation, post-photosynthetic,
sucrose.
Introduction
Stable carbon isotope compositions in tree organs, such as
xylem tissue (tree rings), represent a pivotal tool to examine
changes in plant physiological and metabolic processes as a
response to environmental changes both past and present
(­McCarroll and L­ oader 2004, ­Gessler et al. 2014). Most studies
on δ13C in tree rings are focused on inter-annual and low-­
frequency variations and climatic trends. However, intra-annual
analysis of tree rings and concomitant high temporal resolution
δ13C analysis of different plant compartments provide a better
understanding of the formation of the carbon isotope ratio in
tree rings. Retrospective intra-annual tree ring analyses could
help to decipher environmental conditions more reliably (­Helle
and S
­ chleser 2004, ­Vaganov et al. 2009, ­Schollaen et al.
2013).
­Farquhar et al. (1982) showed that the carbon isotopic signature of newly assimilated photosynthates in the leaves reflects
the interplay of stomatal conductance and photosynthesis, both
responding to environmental conditions such as temperature,
water availability, transpirative demand and light. This signal is
transferred via leaf sucrose through the entire tree and stored in,
e.g., tree rings, which are widely used for palaeoclimate reconstructions (­Kress et al. 2010, ­Hafner et al. 2014). However,
several studies involving intra-annual δ13C analysis have
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Relationship between needle sugars and tree rings 1193
i­ndicated a more complex linkage between tree ring δ13C and
climate due to post-photosynthetic fractionation and carbohydrate storage effects. The latter has been reported to occur particularly early and late in the growing season and especially for
deciduous species (­Helle and S
­ chleser 2004, ­K agawa et al.
2006, ­Skomarkova et al. 2006). Such a carry-over effect may
be seen as the presence of autocorrelation among years hindering climate reconstructions (­Monserud and ­Marshall 2001).
Blurring of the δ13C signal in tree rings has been suggested to
take place also due to mixing of sugar pools of different age
during phloem transport (­Brandes et al. 2006, ­Gessler et al.
2014). Several studies have argued that post-photosynthetic
fractionation processes may be associated with phloem loading
and unloading (­Brandes et al. 2006), occur during xylem cell
formation (­Panek and ­Waring 1997) or occur during respiration
(­Gleixner et al. 1998), altering the climatic signal stored in leaf
sucrose and causing the systematic but varying 13C-enrichment
of sink organs (such as tree rings) in comparison with leaves
(­Gleixner et al. 1993, ­Hill et al. 1995, ­Badeck et al. 2005,
­Gessler et al. 2009b).
Enhanced understanding of isotope fractionation during leaf
level and downstream metabolic processes as well as their
dependency on both environmental and plant physiological factors is a prerequisite for an improved understanding of the formation of the carbon isotope ratio in tree rings. Currently only
few studies have followed the transport of δ13C signal from primary photosynthates to xylem cells at high resolution during a
growing season (­K agawa et al. 2006, ­Brandes et al. 2007,
­Gessler et al. 2009b, ­Offermann et al. 2011). These studies are
based mainly on the δ13C analyses of total extracted leaf carbohydrates or bulk phloem. Hence, they lack information about
individual sugars and the potential fractionation processes of
primary photosynthates occurring at leaf level after photosynthesis and during phloem loading. Our recent study on Siberian
larch (Larix gmelinii Rupr.) needles detected compound-specific
differences in δ13C values of sugars as a response to ambient
weather and enzyme reactions, highlighting the importance of
understanding the leaf level processes at the molecular level for
the development of isotope fractionation transfer models along
the leaf and stem (­Rinne et al. 2015).
Here, we present the first comparison of high-resolution
­compound-specific δ13C data from needle sugars (­Rinne et al.
2015) with intra-annual δ13C measurements from tree rings combined with phenological data. Our study area in Central Siberia is
within the vast boreal forest that plays a critical role in the global
carbon cycle. As a consequence of increasing temperature and
atmospheric CO2, forest growth and subsequently carbon
sequestration may be strongly affected in these ecosystems,
while additionally, permafrost melting could result in the release of
enormous amounts of CO2 and methane (­Schimel et al. 2001).
Understanding the response of boreal forest to changing climate
conditions is, therefore, important. Two contrasting L. gmelinii
f­orest ecosystems on permafrost were studied in two consecutive
growing seasons. The two sites differ markedly in their summer
soil melting depth, which has a strong impact on tree ring growth
(­Kirdyanov et al. 2013, ­Bryukhanova et al. 2015). The main
objectives of this study were to (i) determine the relationship
between needle-level and tree ring isotope signals in larch in
Siberia and (ii) understand how the environmental signal
detected in individual leaf sugars is modified by downstream
metabolic and transport processes and the use of reserves.
Materials and methods
Site description and sampling
The study region near Tura (64°N, 100°E) is in the continuous
permafrost zone of Central Siberia (Russia) and has a typical
continental climate with annual variation of temperature from
−36.8 °C (January) to +15.8 °C (July). The annual precipitation
is only 357 mm, with 171 mm (48%) falling in summer (June–
August). The growing season length is ∼90 days and lasts from
late May or the beginning of June until the beginning of September
(­Bryukhanova et al. 2013). Two sampling sites for L. gmelinii,
which is the dominant tree species in the region, were selected
based on significant differences in growth conditions (air temperature, timing in tree ring development and permafrost depth).
At the TuraH (upper site), which is located at 10 km distance
from and at 434 m higher altitude than the TuraL (lower site)
(589 and 155 m above sea level, respectively), the harsher
growing season conditions are evident in the physical appearance of the trees (narrow radius, sparse crown density). A more
detailed description of the growth conditions can be found in
­Rinne et al. (2015).
In 2011 and 2012, needles were collected between 15 and
19 times from generally four trees per site and season. According to phenological data, the sampling was started at the beginning of or before the start of radial growth, which commenced
at the end of May or in June. The exception is the year 2011 for
TuraL, when the very beginning of the growing season was
missed due to its unusually early start. The last samples were
collected in mid-September, when tree ring formation had
ceased and the needles turned yellow. A more detailed description of the needle sampling can be found in ­Rinne et al. (2015).
For tree ring isotope analyses, two cores were collected at breast
height from six to eight trees per site after the cessation of
growth in 2012.
Xylogenesis observations
Xylogenesis observations were used to link the compound-­
specific sugar results and the laser ablation (LA) tree ring analysis. The same trees were used for defining the xylogenesis
stages as were used for the needle sugar high-performance liquid chromatography (HPLC)–isotope ratio mass spectrometer
(IRMS) analysis with the exception of 2012, when one ­additional
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Tree Physiology Volume 35, 2015
4.38 ± 2.56
4.65 ± 2.81
0.03
0.12
N/A
N/A
0.90*
0.96*
0.03 ± 0.61
0.44 ± 1.06
4.32 ± 1.91
3.81 ± 1.49
−0.10
0.73*
TuraL
TuraH
0.89*
0.91*
1.45 ± 0.74
1.38 ± 0.67
0.98*
0.94*
0.17 ± 0.29
0.44 ± 0.58
δ13C diff.
r-Value
r-Value
r-Value
0.98*
0.95*
1.50 ± 0.50
1.40 ± 0.70
δ13C diff.
δ13C diff.
r-Value
δ13C diff.
δ13C diff.
5 June–15 August
21 August–17 September
r-Value
δ13C diff.
r-Value
Sucrose vs pinitol
Glucose vs
fructose
Sucrose vs glucose
Sucrose vs pinitol
A method modified after ­Wanek et al. (2001) was used for
homogenized needle powder to extract water soluble substances including sugars. A mixture of 60 mg of powder and
1.5 ml of Milli-Q water in Eppendorf tubes was stirred with vortex until the powder was fully suspended. The mixture was then
heated at 85 °C in a water bath for 30 min. Once the samples
were cooled down, they were centrifuged at 10,000g for 2 min.
The supernatant was purified using three types of sample treatment cartridges as described in ­Rinne et al. (2012).
Compound-specific isotope analysis (CSIA) of sugar extracts
was done online using a Delta V Advantage IRMS coupled with
HPLC with a Finnigan LC Isolink interface (­Krummen et al. 2004,
­Rinne et al. 2012). All extracts contained four sugars or sugarlike substances with sufficient concentration for HPLC–IRMS
δ13C analysis: sucrose, glucose, fructose and pinitol/myo-inositol
(from now on referred to as ‘pinitol’). The sugar alcohols pinitol
and myo-inositol cannot be separated, as they have the same
residence time in the analytical column (­Rinne et al. 2012).
A detailed discussion of the environmental signal in the concentration and δ13C values of these sugars can be found in ­Rinne
et al. (2015). The present study will focus on the inter-sugar
Glucose vs fructose
Compound-specific δ 13C measurements
Sucrose vs glucose
Resin and other soluble materials were removed from larch wood
by placing the cores in a Soxhlet apparatus with ethanol for
3 days. The samples were then boiled 6 h in water, which was
replaced every hour, to remove the solvents completely and
dried at 40 °C in an oven for 2 days. Cores were treated with a
microtome to obtain a smooth, even surface for LA-IRMS analysis for the years 2011 and 2012. Following the LA-IRMS analysis, the earlywood (EW) and latewood (LW) fractions of these
years were separated with a scalpel and ground to fine powder
for elemental analyzer (EA)-IRMS measurements.
2012
Preparation of wood cores
2011
tree was used at each site (used also in LA tree ring measurements). Samples (up to 1 cm length) were collected from each
tree using a 5-mm-thick increment borer at 1.3 m height. The
distance between the samples collected per tree was 3 cm. At
TuraL, samples were collected on 20 sampling days in 2011
and on 14 sampling days in 2012. At TuraH, the sampling day
numbers were 14 and 11, respectively. In the laboratory, the
samples were fixed in water–glycerin–ethanol solution (1 : 1 : 1).
Ten-micrometer-thick cross sections were prepared by microtome and stained with safranin (1% solution) and astra blue
(2% solution) for xylogenesis parameter measurements
(­Schweingruber et al. 2006). An optical microscope at ×200
magnification was used to measure the size of different tree ring
zones, number and size of tracheids. Cells in the cambial,
enlargement, wall-thickening and mature zones were differentiated and counted along five radial files.
Table 1. The correlation coefficients and δ13C differences of the sucrose, glucose and fructose δ13C series of TuraL and TuraH. The r-values that are statistically significant (at the 99% level) are
indicated with an asterisk. N/A, not applicable.
1194 Rinne et al.
Relationship between needle sugars and tree rings 1195
δ13C differences (Table 1), which have significance for the intraannual tree ring δ13C values.
In order to prevent isomerization of hexoses (­Rinne et al.
2012), the samples were analysed using low column temperatures. All samples were analysed at 20 °C, which provided chromatographic separation for sucrose, glucose and pinitol. The
samples collected in 2011 were in addition analysed at 15 °C,
which was needed to obtain peak separation for fructose. For
the 2012 samples, the long analytical time was halved by
excluding the analysis at this column temperature. Therefore, the
fructose δ13C and concentration measurements of 2012 are
somewhat affected by partial co-elution with another compound
(­Rinne et al. 2015). For quality control, all samples were analysed at least twice per analytical column temperature. The standard deviation of the repeated analytical measurements per tree
was on an average 0.26‰ for pinitol, 0.37‰ for sucrose,
0.30‰ for glucose and 0.42‰ for fructose.
EA-IRMS δ13C analysis
δ13C
Bulk EW and LW
measurements were made using ∼0.7 mg
of sample, which was weighed into individual tin foil cups and
combusted ‘on-line’ at 1020 °C using an elemental analyser
(EA-1110, Carlo Erba, Milan, Italy) coupled to a DELTA-S IRMS
(Finnigan MAT, Bremen, Germany) via an open split interface
(CONFLO II, Finnigan MAT). The standard deviation for repeated
analysis of reference materials was better than 0.1%.
Intra-annual δ13C analysis
Intra-annual wood δ13C values were determined using an LAcombustion line coupled to an IRMS [ESI/New Wave (Oregon,
USA) UP213 193 nm laser; Sercon (Crewe, UK) CryoPrep coupled to a Sercon IRMS] according to ­Schulze et al. (2004) with
modifications for larger tree ring series as per N.J. Loader et al. (in
preparation). A series of laser shots with 40-μm diameter spot
size (resolution) were sampled as continuous tangential lines
spaced along the same radial line every 40 μm (Figure 1). Care
was taken to avoid visible contaminants, resin ducts, etc. as these
would adversely affect the intra-annual profile sampled at high
resolution. Ablated wood powder was combusted to CO2 over
Cr2O3 at 700 °C and the resulting CO2 cryogenically trapped
prior to gas chromatography and isotope mass spectrometry.
Depending on the ring width, intra-annual resolution consisting of
9–44 data points was consequently obtained for the cores analysed. The samples were run vs in-house reference gas and the
delta values corrected to International Atomic Energy Agency cellulose set at −24.55 per mille International Atomic Energy
Agency. Precision varied a little between runs but is typically
<0.2‰. As these samples were run in ‘small sample’ mode, two
cryo-trapping stages were employed prior to mass spectrometry
in an attempt to maximize sensitivity. Each analysis takes
∼10 min. A standard cellulose paper was also measured by LA,
thereby matching standard and sample preparation. To enable
Figure 1. Picture of annual rings 2011 and 2012 of one of the cores
obtained from Tree 2 in TuraL taken after LA. Resin ducts were avoided
during LA-IRMS analysis, as indicated by the arrows.
comparison between the different laboratories, the LA-IRMS δ13C
values were corrected by 0.77‰ to account for a systematic
offset when the same material was measured with bulk EA-IRMS,
possibly due to an isotope fractionation during LA that differently
affected the cellulose standard and a wood sample.
The presence of residual resins in wood cores after the extraction procedure was studied using attenuated total reflectance
Fourier transform infrared (ATR-FTIR) spectroscopy, as described
in ­Rinne et al. (2005). A System 2000 FTIR spectrometer (Perkin Elmer, USA) Golden Gate single reflection diamond ATR
accessory (Specac, UK) was used. Each spectrum was measured at a spectral resolution of 4 cm−1 and five scans were
taken per sample. The ATR-FTIR analyses indicated that with the
Soxhlet extraction procedure, it was not possible to remove all
resins from all ducts sufficiently. This was seen as the presence
of absorbance band 1690 cm−1 in the infrared spectra (­Rinne
et al. 2005). Care was taken to avoid resin canals during LA
(Figure 1). The cores with high abundance of resin ducts in tree
rings formed in 2011 and 2012 were avoided. Consequently,
the tree selection for the LA-IRMS measurements is not entirely
the same as for the HPLC–IRMS measurements: one (2011) or
two (2012) of the five trees analysed for TuraL and TuraH were
not used for needle sugar analysis.
Meteorological measurements and data analysis
Temperature, vapour pressure deficit (VPD) and precipitation
data at 3-h resolution are available from the meteorological station at Tura (1929–2010), which is located 2 km from TuraL. In
addition, temperature and relative humidity (RH) were measured
both at TuraL and TuraH at hourly resolution. The main difference
between the meteorological data measured directly at the sites
and the data from the station is in the absolute values (during the
sampling seasons, the temperature was higher (TuraL: 0.6 °C,
TuraH: 1.5 °C) and the RH lower (TuraL: 3.9%, TuraH: 0.8%) at
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1196 Rinne et al.
the station) and in the length of the records (no ­site-specific data
are available after 21 August 2012). Hence, since the meteorological data at the sites do not fully cover the sampling season
2012, only the Tura station data are presented (Figure 2).
CurveExpert 1.4 software package was used for wood δ13C
series to determine the best fitting trendlines.
Results
Growing season 2011 and 2012 characteristics
The seasonal course of weather patterns during summer 2011
and 2012 were very different (Figure 2). Year 2011 was exceptional in the sense that the growing season started unusually
early and the warmest conditions occurred at the start of the
growing season followed by a progressive decline. Consequently,
stem expansion started already in late May (Figure 3). A similar
low-frequency trend to that seen in the temperature data is also
present in the VPD record. The period from 25 May until 14 July
is characterized by low precipitation (49 mm) in addition to the
high temperatures. In contrast, the remaining part of the sampling season had high level of precipitation (217 mm by 19
September).
In contrast to 2011, the year 2012 had the typical temperature trend for the region, with the maximum occurring in July
(Figure 2). Again, the VPD series contains the same low-­
frequency trend as temperature. The spring of 2012 was cold
and had the last snow event on 25–28 May. The amount of
precipitation was very low during this growing season until 16
August (total of 48 mm), when the largest single precipitation
event of 2012 occurred (19 mm). The precipitation event was
preceded by a period lasting almost a month having simultaneously the highest temperatures of the month and no rain. Cooler
and wetter (64 mm of rain by 17 September) conditions prevailed during the rest of the sampling season. Stem expansion
started in both TuraL and TuraH on 5 June (Figure 4).
δ 13C needle characteristics
In the following, we concentrate on the sugar δ13C characteristics that are relevant for identifying the mechanisms that underlie the formation of tree ring δ13C values, while a more detailed
description of sugar concentrations and isotope ratios is given
in ­Rinne et al. (2015). In both 2011 and 2012, sucrose,
­glucose and fructose series show common high- and low-­
frequency variability as reflected in highly significant correlations
between individual sugars (Table 1). The δ13C series of sucrose,
the transport sugar that has direct significance for intra-annual tree
ring δ13C variability, are shown separately for all trees in Figures 3
and 4 for TuraL and TuraH. The δ13C results are quite similar
Figure 2. Comparison of VPD (average of 11 am–5 pm), amount of rain as well as temperature (T) at daily resolution in 2011 and 2012. The green
bar indicates the range from Tmin to Tmax. The data are from the Tura meteorological station.
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Relationship between needle sugars and tree rings 1197
Figure 3. Comparison of intra-annual resin extracted wood (series lines and axis titles in black) and needle sucrose (series lines and axis titles in red)
δ13C measurements of each tree for (a) the low elevation site TuraL and (b) TuraH in 2011. Phenophases, consisting of the periods of stem expansion,
lignification and maturation, are given as horizontal lines (green, black and orange) on each group at the top. Periods of earlywood (‘EW’, in yellow)
and latewood (‘LW’, in grey) formation as well as EW/LW, the transition zone between the two stages, are highlighted with background colour. Blue
circles indicate the visual observation of the last LA point on EW.
between the two study sites despite the clear differences in site
conditions and growth of the trees. However, the intra-seasonal
trend of δ13C clearly differs between the 2 years. In 2011, the
δ13C values become increasingly more 13C-depleted from the
beginning of the season until the end of August, whereas a
slight tendency towards more 13C-enriched values is detected
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1198 Rinne et al.
Figure 4. Comparison of intra-annual resin extracted wood (series lines and axis titles in black) and needle sucrose (series lines and axis titles in red)
δ13C measurements of each tree for (a) the low elevation site TuraL and (b) TuraH in 2012. Phenophases, consisting of the periods of stem expansion,
lignification and maturation, are given as horizontal lines (green, black and orange) on each group at the top. Periods of earlywood (‘EW’, in yellow)
and latewood (‘LW’, in grey) formation as well as EW/LW, the transition zone between the two stages, are highlighted with background colour. Blue
circles indicate the visual observation of the last LA point on EW.
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Relationship between needle sugars and tree rings 1199
over the r­emaining period. The opposite low-frequency trends
are seen for 2012.
We then investigated the difference between the δ13C values
of sucrose and needle bulk sugars, since bulk sugar δ13C is
generally used to describe the 13C-depletion of leaves in comparison with sink organs, such as stem (­Gessler et al. 2009a).
We calculated the bulk sugar δ13C values using the concentration and the δ13C data obtained for the four major needle sugars
(­Rinne et al. 2015). The two values were subtracted from each
other for each sampling day (Figure 5). This ‘δ13C difference
sucrose − bulk’ varies strongly, with values between −0.79 and
5.07‰ (average = 2.00 ± 1.08‰), which is mainly due to the
13C-depleted, almost invariable δ13C value of pinitol during the
whole growing season (Figure 6, ­Rinne et al. 2015).
Sucrose δ13C and glucose δ13C values of TuraL and TuraH
show a rather constant offset from each other throughout 2011
and most of 2012 (Table 1, Figure 6). At the end of 2012
(from 21 August), however, this offset disappeared and there is
no longer a significant difference (Table 1). In 2011, glucose is
generally more enriched in 13C than fructose. The 13C-enrichment is rather constant, but the difference is very small and for
all data points is well within the standard deviation (Table 1).
The fructose δ13C values of 2012 cannot be used to define a
similar offset for this year for comparison, because the fructose
chromatographic peak definition in these measurements is
affected by partial co-elution at variable levels with another compound, as discussed in the Materials and methods.
Intra-annual δ 13C analysis of wood cores
The individual wood core δ13C series, which were corrected for
the instrumental 0.77‰ offset, were aligned using an x-axis
that describes the percentage of the annual ring analysed by
each data point, which represent a 40 µm step on the ring
(­Figures 3 and 4). The visual determination of the last laser
shot on EW (prior to LW ablation) is marked with a blue circle
for each series. However, the boundary between EW and LW is
somewhat transitional, and thus, this definition is an approximation. The alignment of the laser δ13C series (x-axis: ‘% of the
ring’) with the sucrose δ13C series (x-axis: ‘sampling day’) containing the phenophases of wood formation (the periods of
wood expansion, lignification and maturation are shown as
horizontal bars) in Figures 3 and 4 was made with the assumption that the δ13C composition of wood is mainly formed during
the expansion and lignification stages. The length of the phenophases was different between the trees, and the selection of
trees for the phenophase and LA-IRMS analysis was not entirely
the same (Figures 3 and 4). Consequently, the aligned wood
δ13C series was treated as a rigid group, whose start and end
points were matched with the observed average dates of the
beginning of EW expansion and the end of LW lignification. For
TuraL, the average day of end of lignification was 15 August
and 22 August in 2011 and 2012, respectively. However, the
extension of the wood δ13C series to these dates would have
meant that some of the blue circles indicating the visual determination of the last laser shot on EW would have been located
on the area indicating the formation time of LW for all trees in
Figures 3a and 4a (‘Latewood (LW)’). Consequently, for TuraL,
the x-axis representing the wood δ13C series (‘% of the ring’)
was somewhat compressed from the right-hand side relative to
the x-axis of sucrose δ13C series (‘sampling day’), so that all
the blue circles were within the EW/LW area (Figures 3a and
4a), which indicates the transition zone between EW and LW
formation for the aligned wood δ13C series.
The similar low-frequency trend in δ13C of sucrose and δ13C
of tree rings over the season, i.e., the general decline in 2011
and increase in 2012, at both sites is striking (Figures 3 and 4,
see Supplementary Data at Tree Physiology Online). The correlation coefficient calculated for the averaged series is 0.76
Figure 5. The δ13C difference between larch needle sucrose and the calculated δ13C value of needle bulk sugar for the sampling days in 2011 and
2012 for TuraL and TuraH.
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1200 Rinne et al.
Figure 6. δ13C values of (a) leaf and stem organic matter of TuraL and TuraH in 2011 and 2012 and (b) their calculated differences. The wood is the
δ13C average measured for the whole series using LA and corrected for the instrumental offset of 0.77‰. The δ13C of needle sucrose is the average
that represents the equivalent period of time as defined in Figures 3 and 4. The needle glucose, fructose and pinitol δ13C values were defined correspondingly. Needle bulk sugar δ13C represents the bulk sugar extract calculated using the δ13C and concentration data of the four main sugars present
in the needle extracts. Sink sucrose δ13C was calculated from needle sucrose δ13C assuming a sole invertase-associated fractionation, when phloem
transported sucrose is hydrolysed for biosynthesis. In addition, three differences between plant organic matter are presented for the two sites and the
2 years. The average difference is also given.
(P < 0.01) and 0.46 (P > 0.01) in 2011 and 0.89 (P < 0.01)
and 0.85 (P < 0.01) in 2012 at TuraL and TuraH, respectively
(Table 1). The slopes of the respective regression lines are 0.07
and 0.02 in 2011 and 0.25 and 0.15 in 2012. At high-­
frequency level, however, a strong dampening is seen in the tree
ring isotope signal, i.e., the smoothed behaviour compared with
the sucrose, indicating that short-term fluctuations in climate
and sucrose δ13C are not reflected in the tree rings but only the
overall trend. The slopes of the regression lines between sucrose
and wood δ13C indicate that <25% of the isotope range
observed in sucrose is visible in the tree rings.
Unlike for the sucrose δ13C series, which all are within a narrow range, some clear offsets occur between the wood δ13C
series representing the individual trees. For TuraL, the Core 1 of
Tree 1 (‘Tree 1_1’ in Figures 3a and 4a) has 13C-depleted values both in 2011 and 2012. The best fit trendline is shown in
Figures 3 and 4 for the average wood δ13C series, which does
not include the Core 1 of Tree 1 for TuraL. For TuraH, a substantial δ13C offset is seen for the wood series of Tree 9 in both years
(Figures 3b and 4b). The offset is 0.64 and 0.96‰ towards
less negative values than the average δ13C series, which does
not include Tree 9, in 2011 and 2012, respectively.
Tree Physiology Volume 35, 2015
The averaged LA-IRMS measurements of resin extracted
wood, which were corrected for the instrumental 0.77‰ offset,
are presented for each site and each year in Figure 6a. To evaluate the δ13C differences between sugars and wood, the average
leaf sucrose δ13C value of the period, when the transported
sugar was consumed for wood formation within a growing season (e.g., Figure 3a: from 25 May to 10 August), was calculated
for each site and year (Figure 6a: ‘needle sucrose’). The δ13C
values of ‘needle bulk sugar’, which represent the weighted
average of the HPLC–IRMS results of the four sugars, were calculated in the similar manner. In Figure 6b, calculated differences
between the δ13C values of sugars and wood are shown for
each site and year. The results show consistent differences
between needle sugars and wood for both years and sites,
where sucrose is more positive and total sugars more negative
than wood.
Discussion
Isotope differences between individual sugars
The strong sucrose 13C-enrichment relative to glucose of ∼1.4‰
over most of the growing season (Table 1, Figure 6) is most
Relationship between needle sugars and tree rings 1201
likely caused by the isotopic selectivity of enzymes that break or
form covalent bonds in glucose metabolism. The most pertinent
reaction of this kind is invertase (­Gilbert et al. 2011), which has
been shown to have a significant role in the regulation of sucrose
metabolism in both developing and mature leaves during a growing season (­Egger and ­Hampp 1993, ­Kingston-Smith et al.
1999, ­Dantas et al. 2005). For yeast (Saccharomyces cerevisiae)
invertase, ­Mauve et al. (2009) reported a ∼1‰ isotope effect
against 13C during invertase catalysis producing 13C-depleted
free fructose and 13C-enriched residual sucrose, which was
assumed to be caused by fractionation at the C-2 position of
fructose. Using 13C nuclear magnetic resonance intramolecular
analysis on source leaf transient starch, grain storage starch and
root storage sucrose, ­Gilbert et al. (2011, ­2012) reported a
similar level of 13C-enrichment of sucrose relative to hexoses to
reflect the combined action of invertase and isomerase.
The 1.4‰ enrichment between sucrose and glucose
observed in the present study (Table 1, Figure 6) suggests that
the invertase-related fractionation is manifested already in leaf
sugars. The relative 13C-enrichment of the transport compound
within leaf can be sustained with continuous synthesis (hexoses) and transport (sucrose) reactions (i.e., branching points)
(­Hobbie and ­Werner 2004). The 0.4‰ bigger enrichment of
sucrose observed in this study compared with the invertaseassociated fractionation for yeast in ­Mauve et al. (2009) may
reflect, for example, the presence of already available residual
remobilized carbohydrates in Tura needles.
The detected 13C-enrichment of sucrose relative to the other
sugars (Figures 5 and 6, Table 1) has implications with regards
to the reported systematic but varying (0–5‰) δ13C difference
between sink organs (stems and roots) (­Schleser 1990, ­1992)
and leaves (­Badeck et al. 2005, ­Gessler et al. 2009a). The
observation that sucrose is the most enriched of the major leaf
sugars could explain the reported δ13C enrichment of phloem,
where sucrose is loaded for downstream transport, relative to
leaves (­Brandes et al. 2006). ­Gessler et al. (2008) found no
fractionation during phloem transport, but further 13C-enrichment of sucrose may occur in sink organs, where sucrose is
broken down for biosynthesis by invertases, which have been
proposed to have an important role in wood formation (­Barratt
et al. 2009, ­Andersson 2014). If a branching point is present
also during wood formation, the kinetic isotope effect of invertase would have an impact on wood δ13C composition, further
affecting the δ13C difference between sink organs and leaves.
This could occur, if, for example, the lighter fraction (glucose and
fructose from invertase) is respired or stored as starch and the
heavier fraction remains for cellulose synthesis.
As an example, on 9 July 2012 at TuraL, the δ13C value of leaf
sucrose was −24.82 ± 0.44‰ (Figure 4). For comparison, the
calculated ‘bulk’ δ13C of leaf sugars on the same sampling day
was −26.79‰. Assuming a sole invertase-associated fractionation on the isotope values in sink organs and isomerization
between glucose and fructose that reaches equilibrium, sucrose
molecules (and cellulose) would inherit a δ13C value of
−24.32‰, which is 0.50 and 2.47‰ more enriched than the
sucrose and bulk sugar δ13C value, respectively, encountered at
the leaf level. Further, any changes in the δ13C difference
between individual leaf sugars from one day to another would
be reflected as a varying bulk sugar δ13C difference between the
leaf and the phloem and would have an impact on the δ13C difference between bulk leaf sugars and a sink organ within the
studied period. As can be seen for 2011 and 2012, for both
study sites, the sucrose to bulk sugar δ13C difference is not stable over time (Figure 5). The main reason for this is the deviant
low- and high-frequency δ13C variability of pinitol/myo-inositol
in comparison with the other sugars (­Rinne et al. 2015):
whereas sucrose, glucose and fructose show generally similar
δ13C variability between each other (Table 1) and respond to
changes in ambient weather conditions with significant changes
in their δ13C value, pinitol δ13C values remained steadily at
around −30‰ throughout each growing season (Table 1).
Another observation possibly related to the varying sucrose to
bulk sugar δ13C difference (Figure 5) is the observed disappearance of the sucrose to glucose general 1.4‰ difference at the
end of the sampling season 2012 (Table 1). A potential mechanism could be a change in the relative activity of invertase and
sucrose synthase (SS). The precise roles of these enzymes in
sucrose breakdown are not fully understood, since both often
appear in the same tissue. For spruce needles, ­Egger and ­Hampp
(1993) reported invertase activity to be higher than that of SS
during the growing season but lower in autumn and winter. Unlike
for invertase, no significant fractionation at the level of the fructose moiety is expected for sucrose catabolism by SS, since the
chemical mechanism in sucrose breakdown does not involve the
cleavage or creation of a C–C bond (­Gilbert et al. 2012). Thus,
any changes in the relative activity of one enzyme over the other
would be reflected in the sucrose to glucose δ13C difference. The
reason for the disappearing difference is intriguing, as this is seen
only in 2012 (Figure 4) but not in 2011 (Figure 3) at both sites,
which would imply that it is not related to the cessation of phenophases and storage processes in autumn nor to cooler and
more humid air at the end of the season (Figure 2) since both
years are similar in this sense. The only major difference between
the 2 years is the sudden release from critical drought conditions,
which followed the main precipitation event of the growing season on 16 August 2012 after a long period with little rain
(­Figure 2, ­Rinne et al. 2015), and which marks the boundary
between the time period with and without the distinct δ13C
enrichment of sucrose over glucose (Table 1). In contrast,
­Ghashghaie et al. (2001) reported similar δ13C values in sucrose
as in glucose and fructose in dehydrated plants, whereas sucrose
13C-enrichment was detected for well-watered individuals. Differences in a varying scale between the three sugars have also been
reported in other studies, but the evaluation and c­ omparison of
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1202 Rinne et al.
these results is hindered by the lack of information on the level of
success of chromatographic baseline separation, which is difficult to achieve for carbohydrates because of their chemical and
physical similarity. For isotopic measurements, the level of peak
separation is of great importance considering the different chromatographic elution times of isotopomers.
The determination of the significance of the small but rather
constant δ13C differences between glucose and fructose seen in
the 2011 data of both sites will require further studies. If the
difference does not occur by chance, it would imply that isomerization between glucose and fructose in plant material does
not fully reach equilibrium. This is supported by the data on
concentrated wine musts presented in ­Guyon et al. (2011),
where an offset of 0.58 ± 0.26‰ (n = 16) can be calculated.
The respective HPLC–IRMS chromatogram indicates good peak
separation for glucose and fructose for accurate δ13C measurements. Unlike for the Tura needle sugars, in ­Guyon et al. (2011),
fructose is more 13C-enriched than glucose, which could be
more characteristic for a sink organ.
Comparison of needle sugar and tree ring δ13C
Many studies have reported that trees significantly rely on carbohydrate reserves during EW formation, particularly broadleaved species, varying from 10 to 75% (­Gäumann 1935,
­Skomarkova et al. 2006, ­Von ­Felten et al. 2007). This was
shown by the application of 14C-labelled photoassimilates
(­Hansen and ­Beck 1990) or inferred from high correlation coefficients between the δ13C of EW and previous year LW (­Switsur
et al. 1995, ­Helle and ­Schleser 2004) and the signal of enriched
leaf starch δ13C in the δ13C signature of EW (­Jäggi et al. 2002).
It could also be inferred from model studies, where a good
agreement between intra-annual δ13C data and modelled values
was found based on a one-pool carbon mixing approach (­Ogée
et al. 2009). With 13C-pulse labelling experiments, ­K agawa et al.
(2006) showed that larch tree saplings growing on a continuous permafrost zone of eastern Siberia were strongly dependent
on photosynthates of the previous year. On the other hand,
­Gessler et al. (2009a) compared bulk phloem sugars and whole
wood of Pinus sylvestris growing in Germany at the resolution of
2 weeks. They found generally a close correspondence in the
seasonal δ13C course between the two carbon pools: an increasing trend for δ13C in the beginning of the growing season lasting
until mid-July followed by a decreasing trend during the remaining part of the growing season.
For Tura, we observed a strong similarity of the low-frequency
trends between the leaf sucrose δ13C series, which are driven by
changes in current weather conditions, particularly VPD (­Rinne
et al. 2015), and the δ13C profiles of annual rings for both years
and sites (Figures 3 and 4, see Supplementary Data at Tree Physiology Online). This suggests that the use of 13C-enriched starch
for EW growth was minimal and that rather current-year climate
conditions govern the changes in isotope ratio of the assimilates
Tree Physiology Volume 35, 2015
and wood. The contradiction to the results of ­K agawa et al.
(2006) for larch in Siberia may be explained by differing carbon
allocation pattern between saplings and mature trees. Further,
the presence of significant amounts of pulse-labelled 13CO2 from
the preceding year in new needles in ­K agawa et al. (2006) can
reflect the abundance of pinitol, which in Tura needles was
observed to have a slow turnover rate and can contribute up to
50% of total soluble carbohydrates (­Rinne et al. 2015).
The relatively small reliance of Tura larch trees on reserves
may be explained by the observation that bud burst usually
occurred before the start of tree ring growth. The independence
of stem growth on starch reserves could also be explained by an
elevated sugar to starch ratio: according to ­Sakai (1966), trees
growing in low temperature environments favour conversion of
starch into sugars or fat, which increase frost hardiness of hardy
cells. Further, if trees in Tura are frequently experiencing water
limitations, they may need more protection to survive the harsh
winter periods provided by the higher sugar to starch ratio
(­Wong et al. 2009). Sugars produced in the fall and stored over
the winter period for, e.g., cryoprotection tend to be more
13C-depleted owing to the cooler weather prevailing during the
time of formation (Figures 3 and 4). Hence, the 13C-depleted
values—relative to the δ13C trend as seen in the leaf sugar
series—detected in the early growing season sequence of the
LA series of 2011, particularly for TuraH, may be explained by
the utilization of these reserves (Figure 3, see Supplementary
Data at Tree Physiology Online). The use of these reserves under
these exceptionally warm and dry conditions prevailing during
the early growing season in 2011 may have been needed due
to the limited availability of freshly produced carbohydrates, particularly when stem growth was at its highest (Figures 2 and 3).
Photoassimilates of larch trees likely reached the stem within
4 days after pulse labelling (­K agawa et al. 2006). The shortterm variations imprinted in Tura sugar δ13C series, however, are
not present in the intra-annual tree ring signal, which is in accordance with the results of ­Gessler et al. (2009a) for P. sylvestris
and of ­Ogée et al. (2009) for Pinus pinaster. This is because the
trunk phloem sap integrates mean photosynthate over several
days (­Keitel et al. 2003) and the wood δ13C in any point reflects
the consecutive sequence of expansion and lignification phenophases (Figures 3 and 4).
The δ13C offset detected for the annual ring 2011 and 2012 of
Tree 1, core 1 of TuraL (Tree 1_1 in Figures 3a and 4a) relative
to the other intra-annual wood series is explained by the variations
in microtopography; the thickness of active soil layer is not similar
below each studied tree. Tree 1 is located closest to the river and
has the fastest rate of seasonal soil thawing: in 2011, the melted
layer reached 30 cm (most of the rooted soil) on 2 June in comparison with between 19 and 26 June for other trees. The other
tree with a significant δ13C offset, Tree 9 of TuraH (Figures 3b and
4b), was found to be rotten inside. Infection of live sapwood with
pathogenic fungi has been connected with large reductions in
Relationship between needle sugars and tree rings 1203
stomatal conductance of the tree (­Mycroft 2010) and hence with
increased δ13C values of wood (­Farquhar et al. 1982).
To compare the δ13C differences between wood and sugars,
the δ13C value of ‘sink sucrose’ was calculated for the Tura data
using the δ13C values of ‘needle sucrose’ (Figure 6a). The δ13C
value of ‘sink sucrose’ was calculated assuming a further invertase-induced fractionation, when phloem transported sucrose is
hydrolysed, and the lighter fraction (glucose and fructose) is
used for respiration or starch formation and the heavier fraction
(‘sink sucrose’) is used for cellulose synthesis, as tentatively
hypothesized above. Assuming an enrichment of wood cellulose
between 1.75 and 2‰—which has the δ13C value of ‘sink
sucrose’ in Figure 6a—relative to Soxhlet-extracted wood
(­Livingston and ­Spittlehouse 1996, ­Sidorova et al. 2010), the
calculated δ13C values of wood (Figure 6a) are on average
0.42‰ more depleted than the δ13C values measured for the
wood samples (Figure 6b: ‘Diff. No. 1’).
The range of the observed δ13C difference between wood and
leaf bulk sugars (Figure 6b: ‘Diff. No. 3’) is well in accordance
with previous studies (­Schleser 1990, ­1992). The ‘Diff. No. 3’
varies from 0.76 to 1.89‰. The results from Tura show that the
significant 13C-depletion of leaf bulk sugars relative to wood is
in large parts due to the high abundance of 13C-depleted pinitol/
myo-inositol in the leaves (­Rinne et al. 2015), as reflected as the
sucrose to bulk sugar δ13C difference in Figure 5. In fact, leaf
sucrose is more enriched (0.95 ± 0.45‰) than resin extracted
wood for both studied sites and years (Figure 6b: ‘Diff. No. 2’).
This level of enrichment is similar to that reported between
phloem sugars and wood tissues of Eucalyptus globulus by
­Cernusak et al. (2005).
(­Helle and ­Schleser 2004, ­Skomarkova et al. 2006, ­Vaganov
et al. 2009). However, we do not understand yet why there was
little use of stored carbohydrates for the studied trees, and the
situation could be different elsewhere.
The comparisons carried out in this study of the leaf sucrose
13
δ C value with that in other sugars and in tree ring elucidated
the causes for the reported 13C-enrichment of sink organs relative to leaves (­Gleixner et al. 1993, ­Badeck et al. 2005, ­Gessler
et al. 2009a). We observed that while needle bulk sugar δ13C
value was on an average 1.2‰ more negative than δ13C value
of Soxhlet-extracted wood, the δ13C value of the transport sugar
sucrose was on average 1.0‰ more positive than that of wood.
This enrichment of sucrose relative to bulk sugars is caused by
the abundance of 13C-depleted pinitol in needle extracts and by
invertase-related fractionation, which produces relative
13C-enrichment of sucrose relative to free hexoses. Further studies applying the combined use of CSIA of sugars (leaf and
phloem) and intra-annual tree ring δ13C analyses are needed for
a broader understanding of the fractionation mechanisms during
the transfer of δ13C signals from leaves to tree rings, to test
whether our findings are also valid for other tree species and
other site conditions. Our study shows a high potential of this
approach for deriving quantitative relationships between climatic
and isotope variability in tree rings. Combined with other methods, such as the application of the dual isotope approach (simultaneous δ13C and δ18O analysis, ­Scheidegger et al. 2000, ­Zech
et al. 2013), the use of CSIA and intra-annual tree ring isotope
analyses will enhance a further mechanistic understanding of
the carbon–water relationships within ecosystems, in particular
for the interpretation of retrospective tree ring analyses.
Conclusions
Supplementary data
Our study provides the first comparison of δ13C values in the
transport sugar sucrose in leaves with δ13C values in tree rings
of larch. It indicates clearly similar low-frequency intra-seasonal
trends between the two records during the expansion and lignification periods of wood, with an approximate 0.8‰ difference
for the two sites and years under investigation. In this study, the
suggested blurring or distortion of the environmental signal
imprinted in leaf sucrose δ13C during tree ring formation, either
due to utilization of starch reserves or due to mixing of sugar
pools of different age during phloem transport (­Brandes et al.
2006, ­K agawa et al. 2006), was detected only to a minor
degree. The latter effect was seen as the subdued amplitude of
short-term δ13C variation in the intra-annual tree ring signal compared with that observed in the sucrose δ13C series. The overall
pattern of climatic variability was retained with little or no sign
of the use of photosynthates formed during the previous
year(s). Consequently, there may be a better chance to reconstruct seasonal climate information from larch trees compared
with other species that have shown dependency on reserves
Supplementary data for this article are available at Tree Physiology
Online.
Acknowledgments
We would like to thank Ineke Lötscher, Lola Schmid and Yves
Letz for their valuable assistance and Rüdiger Kötz for his assistance with ATR-FTIR measurements. Further thanks go to Anatoly Prokushkin, Sergey Tenishev and Sergey Titov for continuous
field work in Tura.
Conflict of interest
None declared.
Funding
This work was supported by Swiss National Science Foundation
(REQIP, 206021_128761 to R.T.W.S., 200021_121838,
Tree Physiology Online at http://www.treephys.oxfordjournals.org
1204 Rinne et al.
200020_134864, 206021_128761), Russian Science Foundation (14-14-00295 to A.V.K. and M.V.B.) and Paul Scherrer
Institute (Instrumental upgrade funds to R.T.W.S.); N.J.L. ack­
nowledges the UK Natural Environment Research Council
(NE/501504), Climate Change Consortium for Wales (C3W)
and European Commission 6th Framework Project ‘Millennium’
(017008).
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