The influence of leaf thickness on the CO2 transfer conductance and leaf stable carbon isotope ratio for some evergreen tree species in Japanese warm-temperate forests

1. The influence of leaf thickness on internal conductance for CO₂ transfer from substomatal cavity to chloroplast stroma ( g _i) and carbon isotope ratio (δ¹³C) of leaf dry matter was investigated for some evergreen tree species from Japanese temperate forests. g _i was estimated based on the combined measurements of gas exchange and concurrent carbon isotope discrimination.
2. Leaves with thicker mesophyll tended to have larger leaf dry mass per area (LMA), larger surface area of mesophyll cells exposed to intercellular air spaces per unit leaf area ( S _mes) and smaller volume ratio of intercellular spaces to the whole mesophyll (mesophyll porosity).
3. g _i of these leaves was correlated positively to S _mes but negatively to mesophyll porosity. The variation in g _i among these species would be therefore primarily determined by variation of the conductance in liquid phase rather than that in gas phase.
4. δ¹³C was positively correlated to mesophyll thickness and leaf nitrogen content on an area basis. However, g _i values did not correlate to δ¹³C. These results suggest that difference in δ¹³C among the species was not caused by the variation in g _i, but mainly by the difference in long-term photosynthetic capacity.
5. Comparison of our results with those of previous studies showed that the correlation between leaf thickness and g _i differed depending on leaf functional types (evergreen, deciduous or annual). Differences in leaf properties among these functional types were discussed.     

Tracing carbon and oxygen isotope signals from newly assimilated sugars in the leaves to the tree-ring archive

The analysis of δ ¹³C and δ ¹⁸O in tree-ring archives offers retrospective insights into environmental conditions and ecophysiological processes. While photosynthetic carbon isotope discrimination and evaporative oxygen isotope enrichment are well understood, we lack information on how the isotope signal is altered by downstream metabolic processes.
In Pinus sylvestris , we traced the isotopic signals from their origin in the leaf water ( δ ¹⁸O) or the newly assimilated carbon ( δ ¹³C), via phloem sugars to the tree-ring, over a time-scale that ranges from hours to a growing season.
Seasonally, variable ¹³C enrichment of sugars related to phloem loading and transport did lead to uncoupling between δ ¹³C in the tree-ring, and the c _i/ c _a ratio at the leaf level. In contrast, the oxygen isotope signal was transferred from the leaf water to the tree-ring with an expected enrichment of 27‰, with time-lags of approximately 2 weeks and with a 40% exchange between organic oxygen and xylem water oxygen during cellulose synthesis.
This integrated overview of the fate of carbon and oxygen isotope signals within the model tree species P. sylvestris provides a novel physiological basis for the interpretation of δ ¹³C and δ ¹⁸O in tree-ring ecology.     

Effect of UV-B radiation on the shoot dry matter production and stable carbon isotope composition of two Arabidopsis thaliana genotypes

An application of stable carbon isotope analysis to the mechanistic interpretation of ultraviolet-B (UV-B) effects on growth inhibition is described that is particularly useful for small plants such as Arabidopsis thaliana that are not well suited for gas exchange studies. Many investigators use tissue δ¹³C, relative abundance of ¹³C and ¹²C, as a proxy for water use efficiency and as an indicator of environmental effects on stomatal behaviour and on photosynthesis during growth. Discrimination against ¹³C is enhanced by both high stomatal conductance and damage to photosynthetic machinery. Because the thinning of the stratospheric ozone layer is permitting more UV-B to enter the biosphere, the mechanisms of action of UV-B radiation on plants are of particular current interest. Arabidopsis thaliana wild-type Landsberg erecta (L er ) and the UV-B-sensitive mutant fah I , deficient in UV-absorbing sinapate esters, were grown in a controlled environment and exposed to UV-B^BE doses of 0 or 6–7 kJ m⁻² day⁻¹. UV-B exposure decreased dry matter production and δ¹³C in both genotypes, but growth inhibition was generally greater in fah I than in L er . The fah I mutant also had less leaf greenness than L er . Changes in leaf tissue δ¹³C were detected before growth inhibition and were evident in treatments of both genotypes that did not cause marked growth effects. This suggests that the effects of UV-B contributing to increased carbon isotope discrimination in L er may have been primarily associated with high stomatal conductance, and in fah I with both high stomatal conductance and damage to photosynthetic machinery.     

Effects of tree height on branch hydraulics, leaf structure and gas exchange in California redwoods

We examined changes in branch hydraulic, leaf structure and gas exchange properties in coast redwood ( Sequoia sempervirens ) and giant sequoia ( Sequoiadendron giganteum ) trees of different sizes. Leaf-specific hydraulic conductivity ( k _L) increased with height in S. sempervirens but not in S. giganteum , while xylem cavitation resistance increased with height in both species. Despite hydraulic adjustments, leaf mass per unit area (LMA) and leaf carbon isotope ratios ( δ ¹³C) increased, and maximum mass-based stomatal conductance ( g _mass) and photosynthesis ( A _mass) decreased with height in both species. As a result, both A _mass and g _mass were negatively correlated with branch hydraulic properties in S. sempervirens and uncorrelated in S. giganteum . In addition, A _mass and g _mass were negatively correlated with LMA in both species, which we attributed to the effects of decreasing leaf internal CO₂ conductance ( g _i). Species-level differences in wood density, LMA and area-based gas exchange capacity constrained other structural and physiological properties, with S. sempervirens exhibiting increased branch water transport efficiency and S. giganteum exhibiting increased leaf-level water-use efficiency with increasing height. Our results reveal different adaptive strategies for the two redwoods that help them compensate for constraints associated with growing taller, and reflect contrasting environmental conditions each species faces in its native habitat.     

Diurnal and seasonal variation in the carbon isotope composition of leaf dark-respired CO2 in velvet mesquite (Prosopis velutina)

We evaluated diurnal and seasonal patterns of carbon isotope composition of leaf dark-respired CO₂ ( δ ¹³C_l) in the C₃ perennial shrub velvet mesquite ( Prosopis velutina ) across flood plain and upland savanna ecosystems in the south-western USA. δ ¹³C_l of darkened leaves increased to maximum values late during daytime periods and declined gradually over night-time periods to minimum values at pre-dawn. The magnitude of the diurnal shift in δ ¹³C_l was strongly influenced by seasonal and habitat-related differences in soil water availability and leaf surface vapour pressure deficit. δ ¹³C_l and the cumulative flux-weighted δ ¹³C value of photosynthates were positively correlated, suggesting that progressive ¹³C enrichment of the CO₂ evolved by darkened leaves during the daytime mainly resulted from short-term changes in photosynthetic ¹³C discrimination and associated shifts in the δ ¹³C signature of primary respiratory substrates. The ¹³C enrichment of dark-respired CO₂ relative to photosynthates across habitats and seasons was 4 to 6‰ at the end of the daytime period (1800 h), but progressively declined to 0‰ by pre-dawn (0300 h). The origin of night-time and daytime variations in δ ¹³C_l is discussed in terms of the carbon source(s) feeding respiration and the drought-induced changes in carbon metabolism.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

A new method to measure carbon isotope composition of CO2 respired by trees: stem CO2 equilibration

1.  Applying Keeling plot techniques to derive δ¹³C of respiratory input in a closed non-equilibrated chamber can lead to large errors because steady-state diffusion rules are violated in a non-steady-state environment. To avoid these errors, respiratory δ¹³C can be derived using equilibrated closed chambers.
2.  We introduce a new method to obtain stem respired CO₂δ¹³C (δ_{st - r}) with closed equilibrated stem chambers (E-SC). We present a theoretical model describing the equilibration process, test the model against field data and find excellent agreement. The method is further tested by comparing it with closed non-equilibrated stem chambers (NE-SC); we found no difference between these methods.
3.  Our theoretical model to describe CO₂ diffusion from the respiratory pool into the chamber and the equation to derive the δ¹³C of the efflux are general. They could be applied to other ecosystem components (e.g. soils).
4.  Our method is easy to implement, cost effective, minimizes sources of error and allows for rigorous leak detection. One major limitation is its inability to detect rapid change; the equilibration process requires 15 ± 2 h. A second limitation is that it cannot be used for species that produce abundant pitch at sites of stem wounding (e.g. Pseudotsuga menziesii ).
5.  Investigating δ¹³C of CO₂ respired by different ecosystem components is necessary to interpret δ¹³C of ecosystem respiration. This parameter has major implications with respect to global carbon cycle science.     

Bulk leaf δ18O and δ13C reflect the intensity of intraspecific competition for water in a semi-arid tussock grassland

We investigated the extent to which plant water and nutrient status are affected by intraspecific competition intensity and microsite quality in a monodominant tussock grassland. Leaf gas exchange and stable isotope measurements were used to assess the water relations of Stipa tenacissima tussocks growing along a gradient of plant cover and soil depth in a semi-arid catchment of Southeast Spain. Stomatal conductance and photosynthetic rate decreased with increasing intensity of competition during the wet growing season, leading to foliar δ ¹⁸O and δ ¹³C enrichment. A high potential for runoff interception by upslope neighbours exerted strong detrimental effects on the water and phosphorus status of downslope S. tenacissima tussocks. Foliar δ ¹⁵N values became more enriched with increasing soil depth. Multiple stepwise regression showed that competition potential and/or rhizosphere soil depth accounted for large proportions of variance in foliar δ ¹³C, δ ¹⁸O and δ ¹⁵N among target tussocks (57, 37 and 64%, respectively). The results presented here highlight the key role that spatial redistribution of resources (water and nutrients) by runoff plays in semi-arid ecosystems. It is concluded that combined measurement of δ ¹³C, δ ¹⁸O and nutrient concentrations in bulk leaf tissue can provide insight into the intensity of competitive interactions occurring in natural plant communities.     

Modelling environmental controls on ecosystem photosynthesis and the carbon isotope composition of ecosystem-respired CO2 in a coastal Douglas-fir forest

We developed and applied an ecosystem-scale model that calculated leaf CO₂ assimilation, stomatal conductance, chloroplast CO₂ concentration and the carbon isotope composition of carbohydrate formed during photosynthesis separately for sunlit and shaded leaves within multiple canopy layers. The ecosystem photosynthesis model was validated by comparison to leaf-level gas exchange measurements and estimates of ecosystem-scale photosynthesis from eddy covariance measurements made in a coastal Douglas-fir forest on Vancouver Island. A good agreement was also observed between modelled and measured δ ¹³C values of ecosystem-respired CO₂ ( δ _R). The modelled δ _R values showed strong responses to variation in photosynthetic photon flux density (PPFD), air temperature, vapour pressure deficit (VPD) and available soil moisture in a manner consistent with leaf-level studies of photosynthetic ¹³C discrimination. Sensitivity tests were conducted to evaluate the effect of (1) changes in the lag between the time of CO₂ fixation and the conversion of organic matter back to CO₂; (2) shifts in the proportion of autotrophic and heterotrophic respiration; (3) isotope fractionation during respiration; and (4) environmentally induced changes in mesophyll conductance, on modelled δ _R values. Our results indicated that δ _R is a good proxy for canopy-level C _c/ C _a and ¹³C discrimination during photosynthetic gas exchange, and therefore has several applications in ecosystem physiology.     

Using stable isotopes to determine soil carbon input differences under ambient and elevated atmospheric CO2 conditions

Quantitative estimates of soil C input under ambient (35 Pa) and elevated (60 Pa) CO₂-partial pressure (pCO₂) were determined in a Free-Air Carbon dioxide Enrichment (FACE) experiment. To facilitate ¹³C-tracing, Trifolium repens L. was grown in a soil with an initial δ¹³C distinct by at least 5‰ from the δ¹³C of T. repens grown under ambient or elevated pCO₂. A shift in δ¹³C of the soil organic C was detected after one growing season. Calculated new soil C inputs in soil under ambient and elevated pCO₂ were 2 and 3 t ha^–1, respectively. Our findings suggest that under elevated CO₂ conditions, soil C sequestration may be altered by changes in plant biomass production and quality.     

Air pollution in the past recorded in width and stable carbon isotope composition of annual growth rings of Douglas-fir

Abstract. Tree-ring indices (TRIs) of annual growth rings in stems of Douglas-fir ( Pseudotsuga menziesii ) growing near a copper smelter showed reduced growth during two multi-year time periods in the past. These periods coincided with World Wars I and II, which are known to represent periods of particularly high SO₂ emissions from the smelter. Reduced growth was correlated with less negative stable carbon isotope composition (δ¹³C) in cellulose purified from wood formed in such years. Based on current models for ¹³C/¹²C in plants, these results indicate that exposure to air pollution resulted in reduced concentration of CO₂ in the intercellular air spaces of the needles. This is consistent with the hypothesis that stomatal closure resulted in impaired photosynthesis and reduced growth during past episodes of high air pollution. The pollution-related change in δ¹³C was superimposed on a change with time in δ¹³C, independent of growth, by - 1.4 per mil from 1902 to 1984.     

δ13C of wood in growth-rings indicates cambial activity of drought-stressed trees of Eucalyptus globulus

1. Growth, density and δ¹³C of wood and leaf area were measured in two adjacent stands of 6 year-old Eucalyptus globulus growing in the 600–700 mm year^–1 rainfall region of south-western Australia. Study sites were identical except for differences in the availability of water owing to physical properties of soil profiles and location of sites within the landscape.
2. Abundance of ¹³C (expressed as δ¹³C) in wood of trees growing on the drought-prone site (– 24·8‰±1·4) was greater than in other trees (– 25·8‰±1·2, P <0·001) throughout the 6 years and, with further development, the δ¹³C signatures of wood may become useful indices of drought-susceptibility in plantations within a few years of establishment. The seasonal pattern of δ¹³C of wood appeared to reflect seasonal variation in water availability and duration of cambial activity.
3. Basic density of wood of trees growing on the more drought-prone site (496±14·0 kg m^–3) was reduced compared to other trees (554±5·3 kg m^–3, P <0·001). δ¹³C of wood across boundaries of growth-rings suggested that drought stopped cambial activity resulting in less production of late wood and less dense wood.
4. The stand growing on the drought-prone site had reduced growth, wood yield and leaf area but identical specific leaf area. Annual growth was correlated with the previous season's rainfall. Together, these results suggested that within the same evaporative climate, drought reduces growth primarily by reducing leaf area and that there is a lag between onset of drought and reduced productivity.     

Population differences in water-use efficiency of Eucalyptus microtheca seedlings under different watering regimes

Gas exchange, water-use efficiency (WUE), carbon isotope composition ( Δ ¹³C) and growth traits were compared among 5 populations of Eucalyptus microtheca F. Muell. Seedlings grown from seed collected across the natural distribution of the species were maintained under water-stressed and well-watered conditions. Gas exchange was measured in terms of net photosynthesis (A) and transpiration (E); WUE was measured in terms of instantaneous water-use efficiency (WUE_i) and transpiration efficiency (WUE_T); growth traits were measured in terms of total biomass (TB), root/shoot ratio (RS), and specific leaf area density (DEN). Significant differences in all traits were detected among the populations. Overall population variation was 1.68–2.50 and 1.48–2.26 μmol CO₂ uptake per mmol H₂O transpired (WUE_i), 1.97–3.04 and 1.64–2.36 g dry matter accumulation per kg water transpired (WUE_T), and Δ ¹³C was −28.81 to −26.75‰ and −30.56 to −30.04‰ under the water-stressed and well-watered conditions, respectively. In addition, WUE_i, WUE_T and Δ ¹³C were significantly correlated with A, E, RS, DEN and TB. The study indicated that measurement of WUE may be a useful trait for selecting genotypes with improved drought adaptation and biomass productivity under different environmental conditions.     

Towards a predictive description of forest canopies from litter properties

1. Specific leaf area (SLA), carbon isotope ratio (δ¹³C) and leaf nitrogen content (N) were measured in litter in 1993 and in the litter layer and canopy in 1996 in a pedunculate Oak ( Quercus robur ) wood at Wytham, Oxford.
2. The frequency distribution of SLA of leaves falling through the late summer and autumn of 1993 changed with a greater contribution of leaves of high SLA earlier in the period. For the same SLA leaves falling earlier in the season had less negative δ¹³C and higher N content than leaves falling later.
3. The frequency distribution of SLA of litter leaves was similar in both years but litter leaves had lower N and less negative δ¹³C in 1996.
4. In the canopy, there was a highly significant negative relationship between height in the canopy and SLA in green and senescent leaves. The relationship between SLA and both δ¹³C ratio and leaf N was different between green, senescent and litter leaves.
5. The frequency distribution of foliage in the canopy estimated from the frequency of SLA in the litter and the vertical pattern of SLA in the canopy was similar to the distribution measured by the 'drop-line' method.     

Fluoride inhibits root water transport and affects leaf expansion and gas exchange in aspen (Populus tremuloides) seedlings

The effects of sodium fluoride (0.3, 5 and 10 m M NaF) on root hydraulic conductivity, and gas exchange processes were examined in aspen ( Populus tremuloides Michx.) seedlings grown in solution culture. A long-term exposure of roots to NaF significantly decreased root hydraulic conductivity ( L _p) and stomatal conductance ( g _s). Root absorbed NaF significantly affected electrolyte leakage in leaf tissues and substantially restricted leaf expansion. NaF did not significantly affect leaf chlorophyll contents but decreased net photosynthesis ( P _n). A short-term exposure of excised roots to 5 m M NaF and KF significantly decreased root water flow ( Q _v) with a concomitant decline in root respiration and reduced g _s when applied through intact roots or excised stems. The same molar concentration of NaCl also decreased Q _v and g _s in intact seedlings, but to a lesser extent than NaF or KF, and did not significantly affect root respiration. The results suggest that fluoride metabolically inhibited Q _v or L _p, probably by affecting water channel activity. We suggest that the metabolic inhibition of L _p by root-absorbed fluoride affected gas exchange and leaf expansion in aspen seedlings.     

Does the 13C of foliage‐respired CO2 and biochemical pools reflect the 13C of recently assimilated carbon?

Isotopic labelling experiments were conducted to assess relationships among ¹³C of recently assimilated carbon ( δC _A), foliage respiration ( δC _F), soluble carbohydrate ( δC _SC), leaf waxes ( δC _LW) and bulk organic matter ( δC _OM). Slash pine, sweetgum and maize were grown under ¹³C depleted CO₂ to label biomass and then placed under ambient conditions to monitor the loss of label. In pine and sweetgum, δC _F of labelled plants (∼−44 and −35‰, respectively) rapidly approached control values but remained depleted by ∼4–6‰ after 3–4 months. For these tree species, no or minimal label was lost from δC _SC, δC _LW and δC _OM during the observation periods. δC _F and δC _SC of labelled maize plants rapidly changed and were indistinguishable from controls after 1 month, while δC _LW and δC _OM more slowly approached control values and remained depleted by 2–6‰. Changes in δC _F in slash pine and sweetgum fit a two-pool exponential model, with the fast turnover metabolic pool (∼3–4 d half-life) constituting only 1–2% of the total. In maize, change in δC _F fits a single pool model with a half-life of 6.4 d. The ¹³C of foliage respiration and biochemical pools reflect temporally integrated values of δC _A, with change in isotopic composition dampened by the size of metabolic carbon reserves and turnover rates.     

Seasonal and annual changes in leaf δ13C in two co-occurring Mediterranean oaks: relations to leaf growth and drought progression

1. Changes of δ¹³C and its relation to leaf development, biochemical content and water stress were monitored over a 2 year period in two co-occurring Mediterranean oak species: the deciduous Quercus pubescens and the evergreen Quercus ilex .
2. The time course of leaf δ¹³C showed different patterns in the two species. Young Q. pubescens leaves had a high δ¹³C and a marked decrease occurred during leaf growth. In contrast, leaves at budburst and maturity did not differ significantly in the case of Q. ilex . We suggest that the difference between δ¹³C of young leaves was linked to differential use of reserves of carbon compounds in the two species.
3. δ¹³C values of mature leaves were negatively correlated with minimum seasonal values of predawn water potential, suggesting that a functional adjustment to water resources occurred.
4. There was a significant correlation between individual δ¹³C values for two successive years. This interannual dependence showed that δ¹³C rankings between trees were constant through time.     

Correlations between net primary productivity and foliar carbon isotope ratio across a Tibetan ecosystem transect

Warming climate could affect leaf-level carbon isotope composition (δ¹³C) through variations in photosynthetic gas exchange. However, it is still unclear to what extent variations in foliar δ¹³C can be used to detect changes in net primary productivity (NPP) because leaf physiology is only one of many determinants of stand productivity. We aim to examine how well site-mean foliar δ¹³C and stand NPP co-vary across large resource gradients using data obtained from the Tibetan Alpine Vegetation Transects (1900–4900 m, TAVT). The TAVT data indicated a robust negative correlation between foliar δ¹³C and NPP across ecosystems (NPP=−2.7224δ¹³C-67.738, r²=0.60, p<0.001). The mean foliar δ¹³C decreased with increasing annual precipitation and its covariation with mean temperature and soil organic carbon and nitrogen contents. The results were further confirmed by global literature data. Pooled δ¹³C data from global literature and this study explained 60% of variations in annual NPP both from TAVT-measures and MODIS-estimates across 67 sites. Our results appear to support a conceptual model relating foliar δ¹³C and nitrogen concentration (N_mass) to NPP, suggesting that: 1) there is a general (negative) relationship between δ¹³C and NPP across different water availability conditions; 2) in water-limited conditions, water availability has greater effects on NPP than N_mass; 3) when water is not limiting, NPP increases with increasing N_mass.     

Relationships of grain δ13C and δ18O with wheat phenology and yield under water-limited conditions

Stable carbon isotope composition (δ¹³C) of dry matter has been widely investigated as a selection tool in cereal breeding programmes. However, reports on the possibilities of using stable oxygen isotope composition (δ¹⁸O) as a yield predictor are very scarce and only in the absence of water stress. Indeed, it remains to be tested whether changes in phenology and stomatal conductance in response to water stress overrule the use of either δ¹³C or δ¹⁸O when water is limited. To answer this question, a set of 24 genotypes of bread wheat ( Triticum aestivum ) were assayed in two trials with different levels of deficit irrigation and a third trial under rainfed conditions in a Mediterranean climate (northwest Syria). Grain yield (GY) and phenology (duration from planting to anthesis and from anthesis to maturity) were recorded, and the δ¹³C and δ¹⁸O of grains were analysed to assess their suitability as GY predictors. Both δ¹³C and δ¹⁸O showed higher broad-sense heritabilities ( H ²) than GY. Genotype means of GY across trials were negatively correlated with δ¹³C, as previously reported, but not with δ¹⁸O. Both isotopes were correlated with grain filling duration, whereas δ¹⁸O was also strongly affected by crop duration from planting to anthesis. We concluded that δ¹⁸O of grains is not a proper physiological trait to breed for suboptimal water conditions, as its variability is almost entirely determined by crop phenology. In contrast, δ¹³C of grains, despite being also affected by phenology, still provides complementary information associated with GY.     

Temporal photosynthetic carbon isotope signatures revealed in a tree ring through 13CO2 pulse-labelling

Using a combined method of pulse-labelling trees and analysing detailed distribution of ¹³C tracer within tree rings, we studied how photo-assimilates incorporated on a given day are then distributed in a tree ring. A branch of a 4-year-old Cryptomeria japonica D.Don tree growing in Tsukuba, Japan was pulse-labelled with non-radioactive ¹³CO₂ on two occasions: 29 May 2001 and 18 September 2001. Two discs were cut from the stem on 4 March 2002, one immediately under and the other 0.5 m below the branch and put through high-resolution δ ¹³C analysis. δ ¹³C peaks were observed in both the earlywood and latewood of the concerned tree ring, corresponding to each pulse-labelling date. The earlywood peaks was broader than the latewood peaks, possibly reflecting seasonal variation of the width of wood developing zone. Half-widths of the peaks were measured and used as indicators for the potential time resolution of tree-ring isotope analysis. The half-widths of the peaks indicated a time resolution no finer than 8.7–28 and 33–42 d in the early and latewood, respectively. Holocellulose extraction yielded only a slight change to the shape of the δ ¹³C peaks. ¹³C tracer pulse-labelled in May and September reached tangentially different locations in the lower disc, suggesting a seasonal change in the pathway of carbohydrates. Local consumption of spring assimilates and long-distance downward transport of autumn assimilates were also suggested.