Beyond CO2-fixation by Rubisco – an interpretation of 13C/12C variations in tree rings from novel intra-seasonal studies on broad-leaf trees

Evidence is presented for a very specific, seasonally recurring tri‐phase carbon isotope pattern in tree rings of broad‐leaf deciduous tree species. It is derived from highly resolved intra‐annual measurements of ¹³C/¹²C ratios of wood and cellulose from tree rings of Fagus sylvatica, Populus nigra, Quercus petraea and Morus alba. Investigations on δ¹³C from buds and leaves of Fagus sylvatica revealed a similar tri‐phase δ¹³C pattern. At the very beginning of a growing season, the δ¹³C trend of tree rings and foliage shows a marked increase of up to 5‰. The maximum δ¹³C‐value of each vegetation period always occurs in young heterotrophic leaves shortly after bud burst and persistently in the early wood of each tree ring, when growth depends on carbon reserves. Thereafter, δ¹³C profiles represent the autotrophic stage of the leaves, which show different patterns of variation, by and large characterized by a decline. The minimum δ¹³C‐value always shows up in the late wood of each tree ring. At the very end of each tree ring δ¹³C‐values start rising again. This increase in δ¹³C marks the gradual switch‐over to storage‐dependent growth and can also be observed in senescent leaves. Seasonal changes of more than 4‰ were measured, whereas contiguous δ¹³C values rarely differed from each other by more than 0.3‰. This tri‐phase pattern cannot be explained by the common model of carbon isotope fractionation during photosynthesis. It appears to be primarily an indication of seasonal changes in down‐stream processes of the carbohydrate metabolism. Environmental influences on the carbon isotope fractionation during photosynthesis are presumably of secondary importance and expressed by certain peculiarities showing up during the autotrophic phase, i.e. the mid‐section of the seasonal δ¹³C pattern.     

Carbon isotope composition of current-year shoots from Fagus sylvatica in relation to growth, respiration and use of reserves

Temporal variations in the stable carbon isotope composition (δ¹³C) of leaves and current‐year stems were examined in beech trees over one year. The δ¹³C of both tissues were equal in the bud stage and started to diverge during growth, with values decreasing by 2·5 and 4·5‰ for stems and leaves, respectively. The dynamics of the δ¹³C and content of non‐structural sugars were also assessed. The beginning of the growth period was characterized by a decrease in starch content and high starch δ¹³C values. Later in the season, the δ¹³C of leaf soluble sugars progressively decreased from the end of May and the δ¹³C of stem sucrose was at least 1·5‰ higher than that of leaves. The δ¹³C of CO₂ respired by stem tissue increased during stem growth and exhibited large seasonal variations ( from ?22·1 to ?26·3‰). These values generally fell between those of starch and total organic matter. The results of the study showed that the δ¹³C of stems is altered by two apparent fractionation steps: one during sugar transfer from leaves to stems and one during stem respiration. These results may have implications for analysis of isotopic signals in tree rings and forest ecosystems.     

Losses of soil carbon by converting tropical forest to plantations: erosion and decomposition estimated by δ13C

Indonesia lost more tropical forest than all of Brazil in 2012, mainly driven by the rubber, oil palm, and timber industries. Nonetheless, the effects of converting forest to oil palm and rubber plantations on soil organic carbon (SOC) stocks remain unclear. We analyzed SOC losses after lowland rainforest conversion to oil palm, intensive rubber, and extensive rubber plantations in Jambi Province on Sumatra Island. The focus was on two processes: (1) erosion and (2) decomposition of soil organic matter. Carbon contents in the Ah horizon under oil palm and rubber plantations were strongly reduced up to 70% and 62%, respectively. The decrease was lower under extensive rubber plantations (41%). On average, converting forest to plantations led to a loss of 10 Mg C ha^?1 after about 15 years of conversion. The C content in the subsoil was similar under the forest and the plantations. We therefore assumed that a shift to higher δ¹³C values in plantation subsoil corresponds to the losses from the upper soil layer by erosion. Erosion was estimated by comparing the δ¹³C profiles in the soils under forest and under plantations. The estimated erosion was the strongest in oil palm (35 ± 8 cm) and rubber (33 ± 10 cm) plantations. The ¹³C enrichment of SOC used as a proxy of its turnover indicates a decrease of SOC decomposition rate in the Ah horizon under oil palm plantations after forest conversion. Nonetheless, based on the lack of C input from litter, we expect further losses of SOC in oil palm plantations, which are a less sustainable land use compared to rubber plantations. We conclude that δ¹³C depth profiles may be a powerful tool to disentangle soil erosion and SOC mineralization after the conversion of natural ecosystems conversion to intensive plantations when soils show gradual increase of δ¹³C values with depth.     

Use of decreasing foliar carbon isotope discrimination during water limitation as a carbon tracer to study whole plant carbon allocation

Foliar carbon isotope discrimination (Δ) of C₃ plants decreases in water‐deficit situations as discrimination by the photosynthetic primary carboxylation reaction decreases. This diminished Δ in leaves under water deficit can be used as a tracer to study whole plant carbon allocation patterns. Carbon isotope composition (δ¹³C value) of leaf hot water extracts or leaf tissue sap represents a short‐term integral of leaf carbon isotope discrimination and thus represents the δ¹³C value of source carbon that may be distributed within a plant in water‐deficit situations. By plotting the δ¹³C values of source carbon against the δ¹³C values of sink tissues, such as roots or stems, it is possible to assess carbon allocation to and incorporation into sink organs in relation to already present biomass. This natural abundance labelling method has been tested in three independent experiments, a one‐year field study with the fruit tree species Ziziphus mauritiana and peach (Prunus persica), a medium‐term drought stress experiment with Ziziphus rotundifolia trees in the glasshouse, and a short‐term drought stress experiment with soybean (Glycine max). The data show that the natural abundance labelling method can be applied to qualitatively assess carbon allocation in drought‐stressed plants. Although it is not possible to estimate exact fluxes of assimilated carbon during water deficit the method represents an easy to use tool to study integrated plant adaptations to drought stress. In addition, it is a less laborious method that can be applied in field studies as well as in controlled experiments, with plants from any developmental stage.     

Short-term variation in the isotopic composition of organic matter allocated from the leaves to the stem of Pinus sylvestris: effects of photosynthetic and postphotosynthetic carbon isotope fractionation

We aimed to quantify the separate effects of photosynthetic and postphotosynthetic carbon isotope discrimination on δ¹³C of the fast‐turn‐over carbon pool (water soluble organic carbon and CO₂ emitted from heterotrophic tissues), including their diel variation, along the pathway of carbon transport from the foliage to the base of the stem. For that purpose, we determined δ¹³C in total and water‐soluble organic matter of the foliage plus δ¹³C and δ¹⁸O in phloem organic matter of twigs and at three heights along the stem of Pinus sylvestris over a nine‐day period, including four measurements per day. These data were related to meteorological and photosynthesis parameters and to the δ¹³C of stem‐emitted CO₂. In the canopy (foliage and twigs), the δ¹³C of soluble organic matter varied diurnally with amplitudes of up to 1.9‰. The greatest ¹³C enrichment was recorded during the night/early morning, indicating a strong influence of starch storage and remobilization on the carbon isotope signatures of sugars exported from the leaves. ¹³C enrichment of soluble organic matter from the leaves to the twig phloem and further on to the phloem of the stem was supposed to be a result of carbon isotope fractionation associated with metabolic processes in the source and sink tissues. CO₂ emitted from the stem was enriched by 2.3–5.2‰ compared with phloem organic matter. When day‐to‐day variation was addressed, water‐soluble leaf δ¹³C and twig phloem δ¹⁸O were strongly influenced by c_i/c_a and stomatal conductance (G_s), respectively. These results show that both photosynthetic and postphotosynthetic carbon isotope fractionation influence δ¹³C of organic matter over time, and over the length of the basipetal transport pathway. Clearly, these influences on the δ¹³C of respired CO₂ must be considered when using the latter for partitioning of ecosystem CO₂ fluxes or when the assessment of δ¹³C in organic matter is applied to estimate environmental effects in c_i/c_a.     

Bicarbonate stimulates non‐structural carbohydrate pools of Camptotheca acuminata

The role of root‐derived dissolved inorganic carbon (DIC) has been emphasized lately, as it can provide an alternative source of carbon for photosynthesis. The fate of newly fixed DIC and its effect on non‐structural carbohydrate (NSC) pools has not been thoroughly elucidated to date. To this end, we used ¹³C (NaHCO₃) as a substrate tracer to investigate the incorporation of newly fixed bicarbonate into the plant organs and NSC compounds of Camptotheca acuminata seedlings for 24 and 72 h. NSC levels across the organs were all markedly increased within 24 h of labeling treatment and afterward only decreased in stems at 72 h. The variation range of NSC concentrations in roots was considerably smaller than in the stem and leaves. As time passed, the δ¹³C in NSC compounds was significantly affected by ¹³C labeling and was more positive in the roots than in the stem and leaves. Starch was more ¹³C‐enriched than was soluble carbohydrate, and the δ¹³C of root starch was as high as ?4.70‰. Bicarbonate incorporation into newly formed NSC compounds contributed up to 0.24% of the root starch within 72 h. These data provided strong evidence that bicarbonate not only acted as a C source that contributed slightly to the NSC pools but also stimulated the increase in NSC pools. The present study expands our understanding of the rapid change of NSC pools across the organs in response to bicarbonate.     

Site‐specific responses to short‐term environmental variation are reflected in leaf and phloem‐sap carbon isotopic abundance of field grown Eucalyptus globulus

The carbon isotopic composition (δ¹³C) of plant material has been used extensively as an indirect measure of carbon fixation per volume of water used. More recently, the δ¹³C of phloem sap (δ¹³C_phl) has been used as a surrogate measure of short‐term, canopy scale δ¹³C. Using a combination of δ¹³C physiological, structural and chemical indices from leaves and phloem sap of Eucalyptus globulus at sites of contrasting water availability, we sought to identify short‐term, canopy scale resource limitations. Results illustrate that δ¹³C_phl offers valid reflections of short‐term, canopy scale values of leaf δ¹³C and tree water status. Under conditions limited by water, leaf and phloem sap photoassimilates differ in ¹³C abundance of a magnitude large enough to significantly influence predictions of water use efficiency. This pattern was not detected among trees with adequate water supply indicating fractionation into heterotrophic tissues that may be sensitive to plant water status. Trees employed a range of physiological, biochemical and structural adaptations to acclimate to resource limitation that differed among sites providing a useful context upon which to interpret patterns in δ¹³C. Our results highlight that such easily characterized properties are ideal for use as minimally invasive tools to monitor growth and resilience of plants to variations in resource availability.     

Environmental and physiological controls on the carbon isotope composition of CO2 respired by leaves and roots of a C3 woody legume (Prosopis velutina) and a C4 perennial grass (Sporobolus wrightii)

Accurate estimates of the δ¹³C value of CO₂ respired from roots (δ¹³C_{R_root}) and leaves (δ¹³C_{R_leaf}) are important for tracing and understanding changes in C fluxes at the ecosystem scale. Yet the mechanisms underlying temporal variation in these isotopic signals are not fully resolved. We measured δ¹³C_{R_leaf}, δ¹³C_{R_root}, and the δ¹³C values and concentrations of glucose and sucrose in leaves and roots in the C₄ grass Sporobolus wrightii and the C₃ tree Prosopis velutina in a savanna ecosystem in southeastern Arizona, USA. Night‐time variation in δ¹³C_{R_leaf} of up to 4.6 ± 0.6‰ in S. wrightii and 3.0 ± 0.6‰ in P. velutina were correlated with shifts in leaf sucrose concentration, but not with changes in δ¹³C values of these respiratory substrates. Strong positive correlations between δ¹³C_{R_root} and root glucose δ¹³C values in P. velutina suggest large diel changes in δ¹³C_{R_root} (were up to 3.9‰) influenced by short‐term changes in δ¹³C of leaf‐derived phloem C. No diel variation in δ¹³C_{R_root} was observed in S. wrightii. Our findings show that short‐term changes in δ¹³C_{R_leaf} and δ¹³C_{R_root} were both related to substrate isotope composition and concentration. Changes in substrate limitation or demand for biosynthesis may largely control short‐term variation in the δ¹³C of respired CO₂ in these species.     

Carbon and oxygen isotope composition of organic compounds in the phloem sap provides a short-term measure for stomatal conductance of European beech (Fagus sylvatica L.)

At eight different dates during the 2000 growing season, δ¹³C and δ¹⁸O were determined in the phloem of adult beech trees growing in natural beech stands in south‐west Germany differing in stand density and local climate. In addition, stand transpiration, precipitation, photosynthetic active radiation, relative air humidity, water pressure deficit of the air, air and soil temperature, soil water potential, and sugar concentration of the phloem sap were determined directly and evaporation and canopy stomatal conductance were modelled. All parameters were related to δ¹³C. The study aimed to identify the time integral within which the δ¹³C of organic compounds transported in the phloem is an indicative measure of these environmental influences. δ¹³C of soluble carbon transported in the phloem was well correlated with mean stomatal conductance in a two‐day integral prior to phloem sampling but did not depend on either light intensity or soil water availability. A strong positive relationship between δ¹³C and δ¹⁸O pointed to observed variation in δ¹³C of phloem sap being a result of variation in stomatal conductance. Bulk leaf δ¹³C was a poor indicator of changes in environmental conditions during the growing season. From these results we conclude that the analysis of δ¹³C in soluble carbon transported in the phloem is a reliable indicator of short‐term changes in C_i/C_a. In contrast, the δ¹³C of structural carbon in beech foliage represents an integration of a range of factors that mask short‐term influences responsible for C_i/C_a.     

Isotope composition of carbon and nitrogen in tissues and organs of <Emphasis Type="Italic">Betula pendula</Emphasis>

Ratios of ¹³С/¹²C and ¹⁵N/¹⁴N isotopes were identified in different parts and organs of drooping birch (Betula pendula Roth) in preforest-steppe and pine-birch forests of the Middle Urals by mass spectrometry. The data were analyzed and interpreted from the perspective of biochemical processes of carbon and nitrogen metabolism in the leaf, cambial tissue, trunk wood, branches, roots, and in the soil. The lighter isotopic composition of carbon is characteristic for the leaves, trunk cambium as well as fine (<2 mm) roots. The trunk wood is characterized by the basal trend for ¹³C enrichment. The heavier carbon isotopic composition inversely related to metabolic activity of organs and tissues, in addition, ¹³С/¹²C ratio corresponds to the nitrogen content in the organs and tissues, indicating the metabolic control of carbon fractionation in woody plants. The isotopic composition of nitrogen in the aboveground parts of the plant (leaves, trunk cambium, wood) and in the medium and fine roots was significantly depleted in ¹⁵N (δ¹⁵N varies from 0 to–3‰), while main roots (δ¹⁵N = 0.6 ‰) and soil (δ¹⁵N = 2.4–6.7‰) were more enriched. The ratio of stable isotopes of carbon and nitrogen is an integrating index of carbon and nitrogen metabolism in plants.     

Pan-European δ13C values of air and organic matter from forest ecosystems

We present carbon stable isotope, δ¹³C, results from air and organic matter samples collected during 98 individual field campaigns across a network of Carboeuroflux forest sites in 2001 (14 sites) and 2002 (16 sites). Using these data, we tested the hypothesis that δ¹³C values derived from large‐scale atmospheric measurements and models, which are routinely used to partition carbon fluxes between land and ocean, and potentially between respiration and photosynthesis on land, are consistent with directly measured ecosystem‐scale δ¹³C values. In this framework, we also tested the potential of δ¹³C in canopy air and plant organic matter to record regional‐scale ecophysiological patterns. Our network estimates for the mean δ¹³C of ecosystem respired CO₂ and the related ‘discrimination’ of ecosystem respiration, δ_er and Δ_er, respectively, were ?25.6±1.9‰ and 17.8 ±2.0‰ in 2001 and ?26.6±1.5‰ and 19.0±1.6‰ in 2002. The results were in close agreement with δ¹³C values derived from regional‐scale atmospheric measurement programs for 2001, but less so in 2002, which had an unusual precipitation pattern. This suggests that regional‐scale atmospheric sampling programs generally capture ecosystem δ¹³C signals over Europe, but may be limited in capturing some of the interannual variations. In 2001, but less so in 2002, there were discernable longitudinal and seasonal trends in δ_er. From west to east, across the network, there was a general enrichment in ¹³C (～3‰ and ～1‰ for the 2 years, respectively) consistent with increasing Gorczynski continentality index for warmer and drier conditions. In 2001 only, seasonal ¹³C enrichment between July and September, followed by depletion in November (from about ?26.0‰ to ?24.5‰ to ?30.0‰), was also observed. In 2001, July and August δ_er values across the network were significantly related to average daytime vapor pressure deficit (VPD), relative humidity (RH), and, to a lesser degree, air temperature (T_a), but not significantly with monthly average precipitation (P_m). In contrast, in 2002 (a much wetter peak season), δ_er was significantly related with T_a, but not significantly with VPD and RH. The important role of plant physiological processes on δ_er in 2001 was emphasized by a relatively rapid turnover (between 1 and 6 days) of assimilated carbon inferred from time‐lag analyses of δ_er vs. meteorological parameters. However, this was not evident in 2002. These analyses also noted corresponding diurnal cycles of δ_er and meteorological parameters in 2001, indicating a rapid transmission of daytime meteorology, via physiological responses, to the δ_er signal during this season. Organic matter δ¹³C results showed progressive ¹³C enrichment from leaves, through stems and roots to soil organic matter, which may be explained by ¹³C fractionation during respiration. This enrichment was species dependent and was prominent in angiosperms but not in gymnosperms. δ¹³C values of organic matter of any of the plant components did not well represent short‐term δ_er values during the seasonal cycle, and could not be used to partition ecosystem respiration into autotrophic and heterotrophic components.     

Moss δ13C: an accurate proxy for past water environments in polar regions

Increased aridity is of global concern. Polar regions provide an opportunity to monitor changes in bioavailable water free of local anthropogenic influences. However, sophisticated proxy measures are needed. We explored the possibility of using stable carbon isotopes in segments of moss as a fine‐scale proxy for past bioavailable water. Variation in δ¹³C with water availability was measured in three species across three peninsulas in the Windmill Islands, East Antarctica and verified using controlled chamber experiments. The δ¹³C from Antarctic mosses accurately recorded long‐term variations in water availability in the field, regardless of location, but significant disparities in δ¹³C between species indicated some make more sensitive proxies. δ¹³C_SUGAR derived from living tissues can change significantly within the span of an Antarctic season (5 weeks) in chambers, but under field conditions, slow growth means that this technique likely represents multiple seasons. δ¹³C_CELLULOSE provides a precise and direct proxy for bioavailable water, allowing reconstructions for coastal Antarctica and potentially other cold regions over past centuries.     

Temporal shift in contribution of terrestrial organic matter to consumer production in a grassland river

1. We used stable isotopes to study the temporal (early summer versus autumn) pattern of use of terrestrial and aquatic sources of organic carbon by consumers in two bedrock‐confined reaches of a grassland river in New Zealand.
2. The major sources of organic carbon available to primary consumers were expected to be terrestrial leaf‐litter and biofilm from the stream channel. These putative carbon sources showed no significant change in mean δ¹³C between summer and autumn. Leaf litter (mean δ¹³C13C compared to biofilm (mean δ¹³C>?19.92).
3. In contrast to leaf litter and biofilm, the δ¹³C of consumers changed over time, being enriched in ¹³C in the autumn compared with early summer. Both the magnitude (>5‰ in some cases) and rapidity of this shift (< 3 months) was surprising.
4. A two‐source mixing model indicated that, during early summer, terrestrial carbon comprised> 50% of tissue carbon for 15 of the 17 taxa of aquatic consumers analysed. During autumn, terrestrial carbon comprised> 50% of the tissue carbon of only five of 25 taxa. Because the mean δ¹³C of putative food sources was consistent over time, the shift in δ¹³C values for consumers is attributed to a change in relative amounts of terrestrial and aquatic carbon available for consumption.
5. Because seston consists of a mixture of many particles of diverse origin, it may provide an integrated measure of catchment‐wide sources of organic matter entering a stream channel. Like the tissues of most consumers, mean δ¹³C values for seston showed a significant shift toward ¹³C enrichment. This indicated that the relative availability of terrestrial carbon decreased from summer to autumn.
6. The actual quantity of carbon contributed to the stream food‐web by this potential terrestrial–aquatic link is unknown. Although terrestrial carbon may comprise a high proportion of the tissue carbon of consumers prior to summer, the majority of secondary production (and carbon sequestration) probably occurs during early summer as a consequence of rising temperature and high quality food in the form of biofilm.     

Carbon autonomy of reproductive shoots of Siberian alder (<Emphasis Type="Italic">Alnus hirsuta</Emphasis> var.<Emphasis Type="Italic"> sibirica</Emphasis>)

Carbon autonomy of current-year shoots in flowering, and of current-year shoots plus 1-year-old shoots (1-year-old shoot system) in fruiting of Siberian alder (Alnus hirsuta var. sibirica) was investigated using a stable isotope of carbon, ¹³C. The current-year shoot and 1-year-old shoot systems were fed ¹³CO₂ and the atom% excess of ¹³C in flowers and fruits was determined. The majority of photosynthate allocated to flower buds was originally assimilated in the leaves of the flowering current-year shoots. Of all the current-year shoots on fruiting 1-year-old shoots, only those nearest to the fruits allocated the assimilated photosynthate to fruit maturation. These results indicate that the current-year shoots and 1-year-old shoot systems are carbon-autonomous units for producing flowers and maturing fruits, respectively.     

Hair of grazing cattle provides an integrated measure of the effects of site conditions and interannual weather variability on δ13C of temperate humid grassland

The carbon isotope composition (δ¹³C) of C3 ecosystems is sensitive to water availability, and provides important information for the assessment of terrestrial carbon (C) sink/source activity. Here, we report the effects of plant available soil water (PAW) on community ¹³C signatures of temperate humid grassland. The 5‐year study was conducted on pastures exhibiting a large range of PAW capacity that were located on two site types: peat and mineral soils. The data set included the centennial drought year 2003, and data from wet years (2000 and 2002). Seasonal variation of PAW was modeled using PAW capacity of each pasture, precipitation inputs and evapotranspiration estimates. Community ¹³C signatures were derived from the δ¹³C of vegetation and segments of tail switch hair of cattle grown while grazing pastures. Hair ¹³C signatures provided an assimilation‐weighted ¹³C signal that integrated both spatial (paddock‐scale) and temporal (grazing season) variation of ¹³C signatures on a pasture. The δ¹³C of hair and vegetation increased with decreasing modeled PAW in the same way on mineral and peat soils. But, at a given PAW, the δ¹³C of hair was 2.6‰ less negative than that of vegetation, reflecting the diet‐hair isotopic shift. Furthermore, the δ¹³C of hair and vegetation on peat soil pastures was 0.5‰ more negative than on pastures situated on mineral soil. This may have resulted from a ～10 ppm CO₂ enrichment of canopy air derived from ongoing peat mineralization. Community‐scale season‐mean ¹³C discrimination (Δ) exhibited a saturation‐type response towards season‐mean modeled PAW (r²=0.78), and ranged between 19.8‰ on soils with low PAW capacity during the drought year of 2003, and 21.4‰ on soils with high PAW capacity in a wet year. This indicated relatively small variation in season‐mean assimilation‐weighted p_i/p_a (0.68–0.75) between contrasting sites and years. However, this range is similar to that reported in other studies, which encompass the range from subtropical arid to humid temperate grassland. Furthermore, the tight relationship between season‐mean Δ and modeled mean PAW suggests that PAW may be used as proxy for Δ.     

Genetic parameters and QTL analysis of δ13C and ring width in maritime pine

Classical quantitative genetics and quantitative trait dissection analysis (QTL) approaches were used in order to investigate the genetic determinism of wood cellulose carbon isotope composition (δ¹³C, a time integrated estimate of water use efficiency) and of diameter growth and their relationship on adult trees (15 years) of a forest tree species (maritime pine). A half diallel experimental set‐up was used to (1) estimate heritabilities for δ¹³C and ring width and (2) to decompose the phenotypic δ¹³C/growth correlation into its genetic and environmental components. Considerable variation was found for δ¹³C (range of over 3‰) and for ring width (range of over 5 mm) and significant heritabilities (narrow sense 0·17/0·19 for δ¹³C and ring width, respectively, 100% additivity). The significant phenotypic correlation between δ¹³C and ring width was not determined by the genetic component, but was attributable to environmental components. Using a genetic linkage map of a full‐sib family, four significant and four suggestive QTLs were detected for δ¹³C, the first for δ¹³C in a forest tree species, as far as known to the authors. Two significant and four suggestive QTLs were found for ring width. No co‐location of QTLs was found between δ¹³C and growth.     

Effects of sampling method on foliar δ13C of Leymus chinensis at different scales

Stable carbon isotope composition (δ¹³C) usually shows a negative relationship with precipitation at a large scale. We hypothesized that sampling method affects foliar δ¹³C and its response pattern to precipitation. We selected 11 sites along a precipitation gradient in Inner Mongolia and collected leaves of Leymus chinensis with five or six replications repeatedly in each site from 2009 to 2011. Additionally, we collected leaves of L. chinensis separately from two types of grassland (grazed and fenced) in 2011. Foliar δ¹³C values of all samples were measured. We compared the patterns that foliar δ¹³C to precipitation among different years or different sample sizes, the differences of foliar δ¹³C between grazed and fenced grassland. Whether actual annual precipitation (AAP) or mean annual precipitation (MAP), it was strongly correlated with foliar δ¹³C every year. Significant difference was found between the slopes of foliar δ¹³C to AAP and MAP every year, among the slopes of foliar δ¹³C to AAP from 2009 to 2011. The more samples used at each site the lower and convergent P‐values of the linear regression test between foliar δ¹³C and precipitation. Furthermore, there was significant lower foliar δ¹³C value in presence of grazed type than fenced type grassland. These findings provide evidence that there is significant effect of sampling method to foliar δ¹³C and its response pattern to precipitation of L. chinensis. Our results have valuable implications in methodology for future field sampling studies.     

The intramolecular ¹³C-distribution in ethanol reveals the influence of the CO₂ -fixation pathway and environmental conditions on the site-specific ¹³C variation in glucose

Efforts to understand the cause of ¹²C versus ¹³C isotope fractionation in plants during photosynthesis and post‐photosynthetic metabolism are frustrated by the lack of data on the intramolecular ¹³C‐distribution in metabolites and its variation with environmental conditions. We have exploited isotopic carbon‐13 nuclear magnetic resonance (¹³C NMR) spectrometry to measure the positional isotope composition (δ¹³C_i, ‰) in ethanol samples from different origins: European wines, liquors and sugars from C₃, C₄ and crassulacean acid metabolism (CAM) plants. In C₃‐ethanol samples, the methylene group was always ¹³C‐enriched (～2‰) relative to the methyl group. In wines, this pattern was correlated with both air temperature and δ¹⁸O of wine water, indicating that water vapour deficit may be a critical defining factor. Furthermore, in C₄‐ethanol, the reverse relationship was observed (methylene‐C relatively ¹³C‐depleted), supporting the concept that photorespiration is the key metabolic process leading to the ¹³C distribution in C₃‐ethanol. By contrast, in CAM‐ethanol, the isotopic pattern was similar to but stronger than C₃‐ethanol, with a relative ¹³C‐enrichment in the methylene‐C of up to 13‰. Plausible causes of this ¹³C‐pattern are briefly discussed. As the intramolecular δ¹³C_i‐values in ethanol reflect that in source glucose, our data point out the crucial impact on the ratio of metabolic pathways sustaining glucose synthesis.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.     

Seasonal dynamics in the stable carbon isotope composition δ¹³C from non-leafy branch, trunk and coarse root CO₂ efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees

Respiration is a substantial driver of carbon (C) flux in forest ecosystems and stable C isotopes provide an excellent tool for its investigation. We studied seasonal dynamics in δ¹³C of CO₂ efflux (δ¹³C_E) from non‐leafy branches, upper and lower trunks and coarse roots of adult trees, comparing deciduous Fagus sylvatica (European beech) with evergreen Picea abies (Norway spruce). In both species, we observed strong and similar seasonal dynamics in the δ¹³C_E of above‐ground plant components, whereas δ¹³C_E of coarse roots was rather stable. During summer, δ¹³C_E of trunks was about ?28.2‰ (Beech) and ?26.8‰ (Spruce). During winter dormancy, δ¹³C_E increased by 5.6–9.1‰. The observed dynamics are likely related to a switch from growth to starch accumulation during fall and remobilization of starch, low TCA cycle activity and accumulation of malate by PEPc during winter. The seasonal δ¹³C_E pattern of branches of Beech and upper trunks of Spruce was less variable, probably because these organs were additionally supplied by winter photosynthesis. In view of our results and pervious studies, we conclude that the pronounced increases in δ¹³C_E of trunks during the winter results from interrupted access to recent photosynthates.