Model for rhizodeposition and CO2 efflux from planted soil and its validation by 14C pulse labelling of ryegrass

A model for rhizodeposition and root respiration was developed and parameterised based on ¹⁴C pulse labelling of Lolium perenne. The plants were grown in a two-compartment chamber on a loamy Haplic Luvisol under controlled laboratory conditions. The dynamics of ¹⁴CO₂ efflux from the soil and ¹⁴C content in shoots, roots, micro-organisms, dissolved organic carbon (DOC) and soil were measured during the first 11 days after labelling. Modelled parameters were estimated by fitting on measured ¹⁴C dynamics in the different pools. The model and the measured ¹⁴C dynamics in all pools corresponded well (r ²=0.977). The model describes well ¹⁴CO₂ efflux from the soil and ¹⁴C dynamics in shoots, roots and soil, but predicts unsatisfactorily the ¹⁴C content in micro-organisms and DOC. The model also allows for division of the total ¹⁴CO₂ efflux from the soil in ¹⁴CO₂ derived from root respiration and ¹⁴CO₂ derived from rhizomicrobial respiration by use of exudates and root residues. Root respiration and rhizomicrobial respiration amounted for 7.6% and 6.0% of total assimilated C, respectively, which accounts for 56% and 44% of root-derived ¹⁴CO₂ efflux from the soil planted with 43-day-old Lolium perenne, respectively. The sensitivity analysis has shown that root respiration rate affected the curve of ¹⁴CO₂ efflux from the soil mainly during the first day after labelling. The changes in the exudation rate influenced the ¹⁴CO₂ efflux later than first 24 h after labelling.     

Pulse-labelling a cover crop with 13C to follow its decomposition in soil under field conditions

A preliminary study was conducted using the stable isotope ¹³C to pulse label the cover crop phacelia (Phacelia tanacetifolia) to examine its decomposition in soil, under field conditions. Plants were grown, in pots, in the greenhouse and after four weeks of growth were labelled with ¹³CO₂ six times, at 1–2 week intervals. A single chamber was placed over the pots, and ¹³CO₂ was generated, inside the chamber, by injecting lactic acid into sodium carbonate (99 atom % ¹³C). For calculating the quantity of Na₂CO₃ required, a target enrichment of 5 atom% ¹³C within the shoots of plants, assuming no respiration losses, was used. When harvested, at flowering, the mean enrichment of the shoot material was 3.0466 atom% ¹³C, or 1.9654 atom% excess ¹³C. To assess uniformity of labelling within plants, the shoot of a single plant was divided into leaves and stem from three sections of equal length. Ninety-three percent of this plant's dry matter had a ¹³C enrichment within 20 % of the weighted mean. At a field site with sandy soil, ¹³C labelled shoot and root material were combined and mixed with soil (0–15 cm). The soil was sampled 16 and 179 days later to determine the recovery of the added excess ¹³C in soil total C. The recoveries in soil (0–30 cm) were, respectively, 78 and 40 % at 16 and 179 days; there was appreciable variation associated with the recovery data from day 16, much less so at day 179. Methodological procedures for (i) enhancing the uniformity of labelling with ¹³C within plants, and (ii) minimising variability in the recovery of ¹³C from soil are suggested. ei]R Merckx     

Carbon balance and allocation of assimilated CO2 in Scots pine, Norway spruce, and Silver birch seedlings determined with gas exchange measurements and 14C pulse labelling

Carbon dioxide is released from the soil to the atmosphere in heterotrophic respiration when the dead organic matter is used for substrates for soil micro-organisms and soil animals. Respiration of roots and mycorrhiza is another major source of carbon dioxide in soil CO₂ efflux. The partitioning of these two fluxes is essential for understanding the carbon balance of forest ecosystems and for modelling the carbon cycle within these ecosystems. In this study, we determined the carbon balance of three common tree species in boreal forest zone, Scots pine, Norway spruce, and Silver birch with gas exchange measurements conducted in laboratory in controlled temperature and light conditions. We also studied the allocation pattern of assimilated carbon with ¹⁴C pulse labelling experiment. The photosynthetic light responses of the tree species were substantially different. The maximum photosynthetic capacity (P _max) was 2.21 μg CO₂ s⁻¹ g⁻¹ in Scots pine, 1.22 μg CO₂ s⁻¹ g⁻¹ in Norway spruce and 3.01 μg CO₂ s⁻¹ g⁻¹ in Silver birch seedlings. According to the pulse labelling experiments, 43–75% of the assimilated carbon remained in the aboveground parts of the seedlings. The amount of carbon allocated to root and rhizosphere respiration was about 9–26%, and the amount of carbon allocated to root and ectomycorrhizal biomass about 13–21% of the total assimilated CO₂. The ¹⁴CO₂ pulse reached the root system within few hours after the labelling and most of the pulse had passed the root system after 48 h. The transport rate of carbon from shoot to roots was fastest in Silver birch seedlings.     

Effects of D-glucose upon D-fuctose metabolism in rat hepatocytes: A 13C NMR study

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-¹³C]fructose, D-[2-¹³C]fructose or D-[6-¹³C]fructose (also 10 mM) in the presence of D₂O. The identification and quantification of ¹³C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of ¹³C-enriched D-fructose, decrease the production of ¹³C-enriched D-glucose, restrict the deuteration of the ¹³C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-¹³C]fructose, and to accentuate the lesser deuteration of the C₂ (as compared to C₅) of ¹³C-enriched D-glucose derived from D-[2-¹³C]fructose. The ratio between C2-deuterated and C₂-hydrogenated L-lactate, as well as the relative amounts of the CH₃-, CH₂D-, CHD₂ and CD₃- isotopomers of ¹³C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C₁ of D-[1-¹³C]glucose generated by hepatocytes exposed to D-[1-¹³C]fructose or D-[6-¹³C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-¹³C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.     

Photosynthesis controls of CO2 efflux from maize rhizosphere

The effects of different shading periods of maize plants on rhizosphere respiration and soil organic matter decomposition were investigated by using a ¹³C natural abundance and ¹⁴C pulse labeling simultaneously. ¹³C was a tracer for total C assimilated by maize during the whole growth period, and ¹⁴C was a tracer for recently assimilated C. CO₂ efflux from bare soil was 4 times less than the total CO₂ efflux from planted soil under normal lighting. Comparing to the normal lighting control (12/12 h day/night), eight days with reduced photosynthesis (12/36 h day/night period) and strongly reduced photosynthesis (12/84 h day/night period) resulted in 39% and 68% decrease of the total CO₂ efflux from soil, respectively. The analysis of ¹³C natural abundance showed that root-derived CO₂ efflux accounted for 82%, 68% and 56% of total CO₂ efflux from the planted soil with normal, prolonged and strongly prolonged night periods, respectively. Clear diurnal dynamics of the total CO₂ efflux from soil with normal day-night period as well as its strong reduction by prolonged night period indicated tight coupling with plant photosynthetic activity. The light-on events after prolonged dark periods led to increases of root-derived and therefore of total CO₂ efflux from soil. Any factor affecting photosynthesis, or substrate supply to roots and rhizosphere microorganisms, is an important determinant of root-derived CO₂ efflux, and thereby, total CO₂ efflux from soils. ¹⁴C labeling of plants before the first light treatment did not show any significant differences in the ¹⁴CO₂ respired in the rhizosphere between different dark periods because the assimilate level in the plants was high. Second labeling, conducted after prolonged night phases, showed higher contribution of recently assimilated C (¹⁴C) to the root-derived CO₂ efflux by shaded plants. Results from ¹³C natural abundance showed that the cultivation of maize on Chromic Luvisol decreased soil organic matter (SOM) mineralization compared to unplanted soil (negative priming effect). A more important finding is the observed tight coupling of the negative rhizosphere effect on SOM decomposition with photosynthesis.     

Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb

Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of ¹³C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C₃ pathway: leaves accumulate organic acids, rich in ¹³C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C₃ pathway: leaves accumulate soluble sugars, poor in ¹³C. After photoperiodic change (long daysshort days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C₃-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20) or C₄-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4) present in the biochemical fractions were calculated from their ¹³C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C₄-origin. Products connected to the C₃ pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C₄ from malate, under long days versus 30 to 70% under short days.Abbreviations CAM crassulacean acid metabolism - CAM pathway pathway with malate accumulation by -carboxylation of PEP, arising from glycolysis of starch (starch-malate sequence) - C₃-metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway (C₃-origin) - C₄-metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway (C₄-origin) - C₃-pathway pathway with RuBPC activity and the Calvin and Benson pathway, irrespective of the CO₂-source, atmospheric or reuptake of the C₄ from malate - ¹³C()=(R_sample-R_PDR)10³/R_PDB where PDB Pee Dee belemnite (belemnite from the Pee Dee formation, South Carolina) and R=¹³C/¹²C - D isotope discrimination - PEP phosphoenolpyruvate - PEPC (EC 4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK (EC 2.7.9.1) pyruvate, Pi-dikinase - RuBP ribulose bisphosphate - RuBPC (EC 4.1.1.39) RuBP carboxylase - SD short days - LD long days     

A re-investigation of the path of carbon in photosynthesis utilizing GC/MS methodology. Unequivocal verification of the participation of octulose phosphates in the pathway

A GC/EIMS/SIM methodology has been developed to re-examine the path of carbon in photosynthesis. Exposing isolated spinach chloroplasts to ¹³CO₂ on a solid support for a defined period followed by quenching and work-up provided a mixture of labelled sugar phosphates. After enzymatic dephosphorylation and derivatization, the Mox-TMS sugars were analysed using the above method. The purpose of the study was to try to calculate the atom% enrichment of ¹³C in as many of the individual carbons in each of the derivatized sugars as was practical using diagnostic fragment ions. In the event, only one 45 s experiment provided sufficient data to enable a range of enrichment values to be calculated. This confirmed that D-glycero-D-altro-octulose phosphate was present in the chloroplasts and was heavily labelled in the C4, C5 and C6 positions, in keeping with the hypothesis that it had an inclusive role and a labelling pattern consistent with a new modified pathway of carbon in photosynthesis.     

Carbon (13C) uptake and allocation in pasture plants following field pulse-labelling

The allocation of carbon (C) to plant roots and conversion to soil organic matter is a major determinant of the size of the terrestrial C pool in pastoral ecosystems. The aim was to quantify C allocation to roots in contrasting pastoral ecosystems. Pastures on long-term research sites in Canterbury, New Zealand were pulse-labelled using ¹³CO₂ within portable gas-tight enclosures. Sites included Winchmore (with or without superphosphate fertiliser, and with or without irrigation) and Tara Hills (low, medium or high grazing intensity with continuous or alternating grazing). Separate micro-plots were labelled in late spring, summer and autumn at Winchmore and in spring at Tara Hills. Herbage label ¹³C recoveries were greatest one hour after pulse labelling and declined by 21 days, whereas in roots they were initially lower but generally continued to increase until 21 days. The greatest recoveries of ¹³C in roots, one hour and 21 days after labelling, were in summer and autumn respectively. The proportion of label ¹³C allocated to roots by 21 days was 0.50 in the absence of superphosphate and 0.41 in the superphosphate treatment, and was 0.39, 0.43 and 0.51 respectively in spring, summer and autumn. Irrigation had no significant effect on root allocation. The low stocking rate at Tara Hills, which had the greatest herbage biomass, also had greater total ¹³C, tussock herbage ¹³C and root ¹³C recoveries than the higher stocking rate treatments. Inter-tussock root recovery and allocation of ¹³C to roots increased with increasing stocking rate, whereas tussock root allocation was greatest in the high and least in the medium stocking rate treatment. By 21 days there was a greater inter-tussock and tussock root recovery and lower inter-tussock herbage recovery in the continuous than in the alternating grazing management treatment. The root allocation was generally greater in the continuous than in alternating grazed treatments, except for tussocks one hour after labelling where the reverse was the case. In conclusion the ¹³C pulse labelling showed pasture plants allocate more C to roots with low soil fertility, high grazing intensity, continuous grazing, and in autumn.     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

13CO2示踪不同化学形态氮素添加对高寒草甸植物光合碳分配的影响

外源氮素(N)输入陆地生态系统后会引起植物和土壤各碳库的变化,但是对不同化学形态氮素的长期输入如何影响光合碳在植物组织、土壤、土壤呼吸中的分配及转运知之甚少,尤其是对于氮输入引起光合碳分配变化进而作用于植物和土壤碳库的机制的认识还非常匮乏。基于在青藏高原矮嵩草草甸开展的不同化学形态氮素添加的长期实验,利用~(13)C示踪方法揭示了光合碳在植物地上、地下组织的分配,及其随时间在土壤中的滞留和随土壤呼吸的释放。研究结果表明,外源氮素添加10年后,与对照未添加氮素处理相比,氨态氮处理下的地上生物量增加了49.5%,氨态氮处理下的地下生物量增加了111.3%。土壤中滞留的~(13)C整体呈下降趋势,氨态氮处理下的土壤碳库显著高于硝态氮处理下的值。不同处理下的土壤呼吸中~(13)C的滞留量随时间呈指数衰减的变化趋势,其中,硝态氮处理下的~(13)C衰减最快。~(13)C同位素标记后第1天测定植物茎和叶内的~(13)C约占刚刚标定完茎和叶内~(13)C的80%,不同处理之间没有显著性差异。直至标记后的第30天,茎和叶内~(13)C的滞留量约占初始量的30%。硝态氮处理下的值在第21天和第30天显著低于对照和氨态氮处理下的值,表明硝态氮处理下,植物光合固定的碳在短期内迅速输入地下组织和土壤中。这些结果从机理上阐明了植物光合碳分配对不同化学形态氮素长期输入的响应,进而影响到土壤呼吸CO_2的释放,以及对土壤碳库动态的贡献。加深了对高寒草甸土壤有机碳库稳定性维持机制的认识,能够为高寒草地的科学管理以及资源的可持续利用提供理论指导。     

Assimilate partitioning affects 13C fractionation of recently assimilated carbon in maize

Coupling ¹³C natural abundance and ¹⁴C pulse labelling enabled us to investigate the dependence of ¹³C fractionation on assimilate partitioning between shoots, roots, exudates, and CO₂ respired by maize roots. The amount of recently assimilated C in these four pools was controlled by three levels of nutrient supply: full nutrient supply (NS), 10 times diluted nutrient supply (DNS), and deionised water (DW). After pulse labelling of maize shoots in a ¹⁴CO₂ atmosphere, ¹⁴C was traced to determine the amounts of recently assimilated C in the four pools and the δ¹³C values of the four pools were measured. Increasing amounts of recently assimilated C in the roots (from 8% to 10% of recovered ¹⁴C in NS and DNS treatments) led to a 0.3‰ ¹³C enrichment from NS to DNS treatments. A further increase of C allocation in the roots (from 10% to 13% of recovered ¹⁴C in DNS and DW treatments) resulted in an additional enrichment of the roots from DNS to DW treatments by 0.3‰. These findings support the hypothesis that ¹³C enrichment in a pool increases with an increasing amount of C transferred into that pool. δ¹³C of CO₂ evolved by root respiration was similar to that of the roots in DNS and DW treatments. However, if the amount of recently assimilated C in root respiration was reduced (NS treatment), the respired CO₂ became 0.7‰ ¹³C depleted compared to roots. Increasing amounts of recently assimilated C in the CO₂ from NS via DNS to DW treatments resulted in a 1.6‰ δ¹³C increase of root respired CO₂ from NS to DW treatments. Thus, for both pools, i.e. roots and root respiration, increasing amounts of recently assimilated C in the pool led to a δ¹³C increase. In DW and DNS plants there was no ¹³C fractionation between roots and exudates. However, high nutrient supply decreased the amount of recently assimilated C in exudates compared to the other two treatments and led to a 5.3‰ ¹³C enrichment in exudates compared to roots. We conclude that ¹³C discrimination between plant pools and within processes such as exudation and root respiration is not constant but strongly depends on the amount of C in the respective pool and on partitioning of recently assimilated C between plant pools. Section Editor: H. Lambers     

Fate of carbon in upland grassland subjected to liming using in situ 13CO2 pulse-labelling

Knowledge of the fate of plant assimilate is fundamental to our understanding of the terrestrial carbon cycle, particularly if we are to predict the effects of changes in climate and land management practices on agroecosystems. Pulse-labelling experiments have revealed that some of the carbon fixed by plants is rapidly allocated below-ground and released back into the atmosphere in respiration. However, little is known about the fate of plant assimilate, not accounted for in soil respiration, in the longer term and how current management practices such as liming may affect this. In southern Scotland, UK, limed and unlimed acid grassland plots were pulse-labelled with ¹³CO₂ and the turnover of ¹³C was studied one and two years after labelling. In this study the amount of labelled carbon remaining in shoot, root, and bulk soil pools, and how this differed between limed and unlimed plots was investigated. The results indicated that plant-root turnover was faster, and plants invested less nitrogen in the roots in the limed plots than in the unlimed plots. More ¹³C remained in the soil in the unlimed treatment compared to the limed treatment, but the main difference was found in the particulate organic matter, which turned over relatively quickly. The label was still above natural abundance one and two years after labelling in many cases. In addition, the results demonstrate that a ¹³CO₂ pulse-label administered for only a few hours can be a useful approach for investigating turnover of carbon several years later.     

Distribution of assimilated carbon in plants and rhizosphere soil of basket willow (Salix viminalis L.)

Willow is often used in bio-energy plantations for its potential to function as a renewable energy source, but knowledge about its effect on soil carbon dynamics is limited. Therefore, we investigated the temporal variation in carbon dynamics in willow, focusing on below-ground allocation and sequestration to soil carbon pools. Basket willow plants (Salix viminalis L.) in their second year of growth were grown in pots in a greenhouse. At five times during the plants growth, namely 0, 1, 2, 3 and 4 months after breaking winter dormancy, a subset of the plants were continuously labelled with ¹⁴CO₂ in an ESPAS growth chamber for 28 days. After the labelling, the plants were harvested and separated into leaves, first and second year stems and roots. The soil was analysed for total C and ¹⁴C content as well as soil microbial biomass. Immediately after breaking dormancy, carbon stored in the first year stems was relocated to developing roots and leaves. Almost half the newly assimilated C was used for leaf development the first month of growth, dropping to below 15% in the older plants. Within the second month of growth, secondary growth of the stem became the largest carbon sink in the system, and remained so for the older age classes. Between 31 and 41% of the recovered ¹⁴C was allocated to below-ground pools. While the fraction of assimilated ¹⁴C in roots and root+soil respiration did not vary with plant age, the amount allocated to soil and soil microbial biomass increased in the older plants, indicating an increasing rhizodeposition. The total amount of soil microbial biomass was 30% larger in the oldest age class than in an unplanted control soil. The results demonstrate a close linkage between photosynthesis and below-ground carbon dynamics. Up to 13% of the microbial biomass consisted of carbon assimilated by the willows within the past 4 weeks, up to 11% of the recovered ¹⁴C was found as soil organic matter.     

Allocation of carbon to shoots,roots, soil and rhizosphere respiration by barrel medic (Medicago truncatula) before and after defoliation

The allocation of carbon to shoots, roots, soil and rhizosphere respiration in barrel medic (Medicago truncatulaGaertn.) before and after defoliation was determined by growing plants in pots in a labelled atmosphere in a growth cabinet. Plants were grown in a ¹⁴CO₂-labelled atmosphere for 30 days, defoliated and then grown in a ¹³CO₂-labelled atmosphere for 19 days. Allocation of ¹⁴C-labelled C to shoots, roots, soil and rhizosphere respiration was determined before defoliation and the allocation of ¹⁴C and ¹³C was determined for the period after defoliation. Before defoliation, 38.4% of assimilated C was allocated below ground, whereas after defoliation it was 19.9%. Over the entire length of the experiment, the proportion of net assimilated carbon allocated below ground was 30.3%. Of this, 46% was found in the roots, 22% in the soil and 32% was recovered as rhizosphere respiration. There was no net translocation of assimilate from roots to new shoot tissue after defoliation, indicating that all new shoot growth arose from above-ground stores and newly assimilated carbon. The rate of rhizosphere respiration decreased immediately after defoliation, but after 8 days, was at comparable levels to those before defoliation. It was not until 14 days after defoliation that the amount of respiration from newly assimilated C (¹³C) exceeded that of C assimilated before defoliation (¹⁴C). This revised version was published online in June 2006 with corrections to the Cover Date.     

Losses of14C from roots of pulse-labelled wheat and barley during washing from soil

In crop carbon budget studies losses of root material during storage and washing of samples may cause considerable errors. To correct data from field experiments where rhizosphere C fluxes in wheat and barley were determined by¹⁴C pulse-labelling at different development stages, experiments were performed to quantify losses of¹⁴C from roots during washing. Losses of¹⁴C from wheat roots grown on nutrient solution and stored in different ways, decreased from on average 45% of total¹⁴C content 8 days after labelling to 27% after 21 days. This decrease was probably related to the incorporation of¹⁴C into structural compounds. During washing of oven-dried soil cores of held-grown wheat and barley 3 weeks after labelling, different size classes of losses of¹⁴C from the roots increased substantially with the development stage of the crop at labelling. The 0.3–0.6 mm size class increased from 5% of the¹⁴C in roots > 0.3 mm in young plants to 25% at ripening, and the < 0.3 mm size class increased from 8 to 41% of total¹⁴C content. The latter size class was, however, determined by washing handpicked roots and may therefore partly consist of adhering exudates, mucilages and microorganisms. The effect of development stage on root washing losses was attributed to root senescence which increases the fragility of roots. Thus, especially at the rate development stages root washing losses caused a severe underestimation of the root¹⁴C content. However, with these results the¹⁴C distribution patterns of the field experiments could be adequately corrected.Communication No. 77 of the Dutch Programme on Soil Ecology of Arable Farming Systems.     

Simultaneous CT-13C and VT-15N chemical shift labelling: Application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH

Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) ¹³C and variable time (VT) ¹⁵N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J_CC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2H_zN_z state, then prior to the VT chemical shift labelling period is converted into 2H_zN_y coherence. The VT -¹⁵N mode eliminates the effect of ¹ J _N,CO and ^1,2 J _N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH₂ groups, thus removing any potential overlap with the CH₃ signals. The CT-¹³CH₃,VT-¹⁵N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH₃NH and 3D ¹³C,¹⁵N HSQC-NOESY-CH₃NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH₃ and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH₃ and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.     

Scope for use of stable carbon isotopes in discerning the incorporation of forest detritus into aquatic foodwebs

Stable isotope analysis of carbon has been proposed as a means for discerning the incorporation of terrestrial forest detritus into aquatic foodwebs, and as such, has the potential to be used as a biomonitor of the aquatic effects of riparian deforestation. A synthesis of ¹³C/¹²C data from the literature indicates, however, that the scope for successful use of carbon isotope analysis in separating allochthonous and autochthonous food provenance is much more limited than was once thought. This occurs due the overlap in carbon isotope ratios between terrestrial forest detritus and those of both lotic attached algae and lentic filamentous attached algae. Only within rockyshored, oligotrophic lakes without macrophytes, and forest-fringed estuaries and lagoons, where the carbon isotope ratios for attached algae and forest detritus are significantly different, is there any likelihood of discerning the incorporation of allochthonous carbon into aquatic foodwebs using ¹³C/¹²C values alone.     

Evaluation of the wick method for in situ 13C and 15N labelling of annual plants using sugar-urea mixtures

To investigate the amount and fate of root-derived C and N, often tracer techniques are used, where plants are labelled with isotopes. In the present study, we evaluated the suitability of the cotton wick method for in situ labelling of peas (Pisum sativum L.) and oats (Avena sativa L.) with ¹³C and ¹⁵N simultaneously. With two greenhouse experiments we investigated how the wick method and aqueous urea and sugar solutions at a variety of concentrations affected plant development. In addition, we investigated the distribution of ¹³C and ¹⁵N in plants from column experiments under outdoor conditions. Solution was taken up by the plant from a small vial connected to the stem by a cotton wick which was passed through a hole in the stem of the plants. Generally, solution uptake varied between individual plants and decreased with increasing sugar concentrations. Below-ground, above-ground and total plant dry matter, were not significantly affected by the wick method and the applied solutions. Mixtures of aqueous glucose solutions at 2 to 4% and aqueous urea solutions at 1% are useful carriers of ¹³C and ¹⁵N. However, in the investigated plants isotopes were not homogeneously distributed among plant parts. Above-ground plant biomass was preferentially enriched with ¹³C and ¹⁵N, whereas below-ground plant biomass was generally lower enriched. Moreover, isotope distribution ratio of individual plants varied considerably, independent of plant part or timing of labelling. This must be taken into account when estimating root-derived C and N. Future studies comparing labelling methods need to present the isotope distribution ratios among plant parts to allow a true comparison of the methods and the evaluation of their suitability for estimating rhizodeposition.     

Comparisons of δ<Superscript>13</Superscript>C of photosynthetic products and ecosystem respiratory CO<Subscript>2</Subscript> and their responses to seasonal climate variability

This study investigated the relationship between ¹³C of ecosystem components, soluble plant carbohydrates and the isotopic signature of ecosystem respired CO₂ (¹³C_R) during seasonal changes in soil and atmospheric moisture in a beech (Fagus sylvatica L.) forest in the central Apennine mountains, Italy. Decrease in soil moisture and increase in air vapour pressure deficit during summer correlated with substantial increase in ¹³C of leaf and phloem sap soluble sugars. Increases in ¹³C of ecosystem respired CO₂ were linearly related to increases in phloem sugar ¹³C (r²=0.99, P0.001) and leaf sugar ¹³C (r²=0.981, P0.01), indicating that a major proportion of ecosystem respired CO₂ was derived from recent assimilates. The slopes of the best-fit lines differed significantly (P0.05), however, and were about 0.86 (SE=0.04) for phloem sugars and about 1.63 (SE=0.16) for leaf sugars. Hence, changes in isotopic signature in phloem sugars were transferred to ecosystem respiration in the beech forest, while leaf sugars, with relatively small seasonal changes in ¹³C, must have a slower turnover rate or a significant storage component. No significant variation in ¹³C was observed in bulk dry matter of various plant and ecosystem components (including leaves, bark, wood, litter and soil organics). The apparent coupling between the ¹³C of soluble sugars and ecosystem respiration was associated with large apparent isotopic disequilibria. Values of ¹³C_R were consistently more depleted by about 4 relative to phloem sugars, and by about 2 compared to leaf sugars. Since no combination of the measured pools could produce the observed ¹³C_R signal over the entire season, a significant isotopic discrimination against ¹³C might be associated with short-term ecosystem respiration. However, these differences might also be explained by substantial contributions of other not measured carbon pools (e.g., lipids) to ecosystem respiration or contributions linked to differences in footprint area between Keeling plots and carbohydrate sampling. Linking the seasonal and inter-annual variations in carbon isotope composition of carbohydrates and respiratory CO₂ should be applicable in carbon cycle models and help the understanding of inter-annual variation in biospheric sink strength.     

The heterogeneous nature of microbial products as shown by solid-state13C CP/MAS NMR spectroscopy

Homoionic Na-, Ca-, and Al-clays were prepared from the <2 m fractions of Georgia kaolinite and Wyoming bentonite and mixed with sand to give artificial soils with 5, and 25% clay. The artificial soils were inoculated with microbes from a natural soil before incubation. Unlabelled and uniformly¹³C-labelled (99.9% atom) glucose were incorporated into the artificial soils to study the effects of clay types, exchangeable cations and clay contents on the mineralization of glucose-carbon and glucose-derived organic materials. Chemical transformation of glucose-carbon upon incorporation into microbial products and metabolites, was followed using solid-state¹³C CP/MAS NMR spectroscopy.There was a significant influence of exchangeable cations on the mineralization of glucose-carbon over a period of 33 days. At 25% clay content, mineralization of glucose-carbon was highest in Ca-soils and lowest in Al-soils. The influence of exchangeable cations on mineralization of glucose-carbon was more pronounced in soils with bentonite clay than those with kaolinite clay. Statistical analysis of data showed no overall effect of clay type on mineralization of glucose-carbon. However, the interactions of clay type with clay content and clay type with clay content and exchangeable cations were highly significant. At 25% clay content, the mineralization of glucose-carbon was significantly lower in Na- and Al-soils with Wyoming bentonite compared with Na- and Al-soils with Georgia kaolinite. For Ca-soils this difference was not significant. Due to the increased osmotic tension induced by the added glucose, mineralization of glucose-carbon was slower in soils with 5% clay than soils with 25% clay.Despite the differences in the chemical and physical characteristics of soils with Ca-, Na- and Al-clays, the chemical composition of organic materials synthesised in these soils were similar in nature. Assuming CP/MAS is quantitative, incorporation of uniformly¹³C-labelled glucose (99.9% atom) in these soils resulted in distribution of carbon in alkyl (24–25%), O-alkyl (56–63%), carbonyl (11–15%) and small amounts of aromatic and olefinic carbon (2–4%). However, as decomposition proceeded, the chemistry of synthesised material showed some changes with time. In the Ca- and Na-soils, the proportions of alkyl and carbonyl carbon decreased and that of O-alkyl carbon increased with time of incubation. However, the opposite trend was found for the Al-soil.Proton-spin relaxation editing (PSRE) subspectra clearly showed heterogeneity within the microbial products. Subspectra of the slowly-relaxing (long T₁(H)) domains were dominated by alkyl carbon in long- and short-chain structures. The signals due to N-alkyl (55 ppm) and carbonyl carbon were also strong in these subspectra. These subspectra were very similar to those obtained for microbial and fungal materials and were probably microbial tissues attached to clay surfaces by polysaccharide extracellular mucilage. Subspectra of fast-relaxing (short T₁(H)) domains comprised mostly O-alkyl and carbonyl carbon and were probably microbial metabolites released as neutral and acidic sugars into the extracellular environment, and strongly sorbed by clay surfaces.