Summary• Half of the biological activity in forest soils is supported by recent tree photosynthate, but no study has traced in detail this flux of carbon from the canopy to soil microorganisms in the field.• Using 13 CO 2 , we pulse-labelled over 1.5 h a 50-m 2 patch of 4-m-tall boreal Pinus sylvestris forest in a 200-m 3 chamber.• Tracer levels peaked after 24 h in soluble carbohydrates in the phloem at a height of 0.3 m, after 2-4 d in soil respiratory efflux, after 4-7 d in ectomycorrhizal roots, and after 2-4 d in soil microbial cytoplasm. Carbon in the active pool in needles, in soluble carbohydrates in phloem and in soil respiratory efflux had half-lives of 22, 17 and 35 h, respectively. Carbon in soil microbial cytoplasm had a half-life of 280 h, while the carbon in ectomycorrhizal root tips turned over much more slowly. Simultaneous labelling of the soil with showed that the ectomycorrhizal roots, which were the strongest sinks for photosynthate, were also the most active sinks for soil nitrogen.• These observations highlight the close temporal coupling between tree canopy photosynthesis and a significant fraction of soil activity in forests.

Changes in the 13C discrimination of current leaf photosynthesis might have profound impacts on root respiratory substrates. Therefore, the aim of this study was (1) to refine a method for the isolation of root and leaf starch and soluble sugars (neutral fraction) for stable carbon isotope analysis and (2) to assess the short-term temporal variability of the C isotope composition (delta13C) of starch and of the neutral fraction of beech roots and leaves at different canopy heights. An existing method for isolating starch for stable C isotope analysis based on enzymatic hydrolysis was modified to account for the low starch content of the samples. This was achieved by removing the enzyme (alpha-amylase) by ultrafiltration after the hydrolysis, resulting in very low carbon blanks. The neutral fraction was separated from organic acids and cations by ion-exchange chromatography. An anion-exchange resin in the [HCO3]--form was chosen that ensured high precision of C blanks. Beech leaves at 5, 10 and 20 m above the forest floor as well as roots were sampled six times during a day/night cycle in July 2003. Delta13C values of bulk material, starch and the neutral fraction increased from the lower to the higher canopy with mean differences between 5 and 20 m of 3.8, 3.4 and 2.7 per thousand for the delta13C values of starch, neutral fraction and bulk foliage, respectively. The delta13C value of foliar starch increased from the morning to the afternoon and decreased during the night, but diurnal differences (up to 3.1 per thousand) were only statistically significant for leaves sampled at 5 and 10 m height. In roots, no diurnal variation in the delta13C of starch was observed during the short time frame of one day and the delta13C of the neutral fraction did not differ between samples taken at 16:30 and 22:00. Calculated delta13C values of starch, which was mobilised during the night, were more positive than the total starch (all sampling times pooled) in leaves. Furthermore, the delta13C values of mobilised starch were approximately 5 per thousand more positive than that of the mobilised neutral fraction. Hence, the delta13C of potential sources for export from canopy leaves to roots varied considerably in their C isotope composition.

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

Characterization of soil respiration rates and delta(13)C values of soil-respired CO(2) are often based on measurements at a particular time of day. A study by Gower et al. (2001) in a boreal forest demonstrated diurnal patterns of soil CO(2) flux using transparent measurement chambers that included the understory vegetation. It is unclear whether these diurnal patterns were solely the result of photosynthetic CO(2) uptake during the day by the understory or whether there were underlying trends in soil respiration, perhaps driven by plant root allocation, as recently demonstrated in Mediterranean oak savannah. We undertook intensive sampling campaigns in a boreal Picea abies L. Karst. forest to investigate whether diurnal variations in soil respiration rate and stable carbon isotope ratio (delta(13)C) exist in this ecosystem when no understory vegetation is present in the measurement chamber. Soil respiration rates and delta(13)C were measured on plots in which trees were either girdled (to terminate the fraction of soil respiration directly dependent on recent photosynthate from the trees), or not girdled, every 4 h over two 48-hour cycles during the growth season of 2004. Shoot photosynthesis and environmental parameters were measured concurrently. No diurnal patterns in soil respiration rates and delta(13)C were observed in either treatment, despite substantial variations in climatic conditions and shoot photosynthetic rates in non-girdled trees. Consequently, assessment of daily soil respiration rates and delta(13)C in boreal forest systems by single, instantaneous daily measurements does not appear to be confounded by substantial diurnal variation.

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