Rhizosphere carbon flow measurement and implications: from isotopes to reporter genes

Despite the fundamental importance of rhizosphere C-flow in managed and natural systems, reliable measurement/resolution of C-flow and assessment of its consequences have largely remained elusive to soil biologists. Techniques involving both radioactive (¹⁴C) and stable (¹³C) isotopes of carbon have made some progress in terms of studying rhizosphere C-flow. Pulse-chase techniques have been used effectively to study dynamics of C-transfer to the rhizosphere and rhizosphere microbial biomass. The information obtained through pulse-chase is strongly dependent on the chase period following the labelling event. Continuous labelling is primarily used to determine plant inputs to soil over an extended time period and includes all kinds of C input – from root turnover, root respiration, root exudation, production of mucilage, etc. One of the main constraints to both approaches is that distinguishing root from microbial respiration is difficult, if not impossible. ¹³C techniques have gone some way towards resolving this difficulty, although ¹³C signatures in the plant–soil system are not easy to interpret and detailed resolution of carbon flow through different components of the rhizosphere biomass is unlikely to be achieved in such an inherently `noisy' system. Recent developments in molecular biology now provide a new opportunity to resolve rhizosphere C-flow and its implications. Reporter gene systems where, for example, rhizobacteria are marked with lux and unstable gfp reporters, overcome the difficulty of distinguishing root and microbial C fluxes and complement the isotopic and more traditional approaches. Reporter systems have now begun to resolve the competitive C sink strengths of different components of the rhizosphere microbial community and assess how a rhizobacterial inoculum may change C-flow in applications such as disease control and rhizoremediation of contaminated land. Fusion of reporter genes to nutrient (N and P) starvation genes in rhizobacteria has also enabled in situ characterisation of nutrient depletion around the root and assessment of the impact of changes in C-flow (such as those induced by climate change) on nutrient depletion dynamics. The availability of an integrated approach involving isotopic, molecular biological and other techniques now offers an exciting new era where reliable measurement and resolution of rhizosphere C-flow (and its consequences) can contribute to our understanding of ecosystem function and to management of crop-microbe interactions.     

A continuous labelling approach to recover photosynthetically fixed carbon in plant tissue and rhizosphere organisms of young beech trees (Fagus sylvatica L.) using 13C depleted CO2

A continuous labelling experiment using ¹³C-CO₂ was set up in open-top chambers in order to follow fluxes of assimilates from the plant into the rhizosphere. Labelling was performed for one growing season by adding low amounts of CO₂ depleted in ¹³C to the atmosphere of the open-top chambers, resulting in a difference of ? ¹³C 5‰ V-PDB compared to ambient conditions. The label was recovered in both plant parts and soil microbial communities, analysed via phospholipid fatty acid (PLFA) side chains. PLFA 18:2ω6,9 showed a significant incorporation of the ¹³C label in October, indicating that fungi utilized plant derived carbon. In bacterial PLFA no label incorporation was detected, probably due to a lower use of rhizodeposits or a preference to older carbon compounds as energy sources. This experimental setup represents a low-cost continuous labelling method for field experiments with only minor increase of CO₂ concentrations.     

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.     

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.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Biotic controls on CO2 and CH4 exchange in wetlands – a closed environment study

Wetlands are significant sources of the important greenhouse gas CH₄. Here we explore the use of an experimental system developed for the determination of continuous fluxes of CO₂ and CH₄ in closed ecosystem monoliths including the capture of ¹⁴CO₂ and ¹⁴CH₄ following pulse labelling with ¹⁴CO₂. We show that, in the ecosystem studied, ebullition (bubble emission) may account for 18 to 50% of the total CH₄ emission, representing fluxes that have been difficult to estimate accurately in the past. Furthermore, using plant removal and ¹⁴C labelling techniques, we use the system to detail the direct influence of vascular plants on CH₄ emission. This influence is observed to be dependent on the amount of vascular plants present. The results that may be produced using the presented experimental set-up have implications for an improved understanding of wetland ecosystem/atmosphere interactions, including possible feedback effects on climate change. In recent years much attention has been devoted to ascertaining and subsequently using the relationship between net ecosystem productivity and CH₄ emission as a basis for extrapolation of fluxes across large areas. The experimental system presented may be used to study the complex relationship between vascular plants and CH₄ emission and here we show examples of how this may vary considerably in nature between and even within ecosystems.     

Fate and dynamics of recently fixed C in pasture plant–soil system under field conditions

The flow of photosynthetically fixed C from plants to selected soil C pools was studied after ¹³CO₂ pulse labeling of pasture plants under field conditions, dynamics of root-derived C in soil was assessed and turnover times of the soil C pools were estimated. The transport of the fixed C from shoots to the roots and into the soil was very fast. During 27 h, net C belowground allocation reached more than 10% of the fixed C and most of the C was already found in soil. Soil microbial biomass (C_MIC) was the major sink of the fixed C within soil C pools (ca 40–70% of soil ¹³C depending on sampling time). Significant amounts of ¹³C were also found in other labile soil C pools connected with microbial activity, in soluble organic C and C associated with microbial biomass (hot-water extract from the soil residue after chloroform fumigation-extraction) and the ¹³C dynamics of all these pools followed that of the shoots. When the labelling (2 h) finished, the fixed ¹³C was exponentially lost from the plant–soil system. The loss had two phases; the first rapid phase corresponded to the immediate respiration of ¹³C during the first 24 h and the second slower loss was attributable to the turnover of ¹³C assimilated in C_MIC. The corresponding turnover times for C_MIC were 1.1 days and 3.4 days respectively. Such short turnover times are comparable to those measured by growth kinetics after the substrate amendment in other studies, which indicates that microbial growth in the rhizosphere is probably not limited by substrate availability. Our results further confirmed the main role of the soil microbial community in the transformation of recently fixed C, short turnover time of the easily degradable C in the rhizosphere, and its negligible contribution to more stable soil C storage.     

Root and microbial involvement in the kinetics of 14C-partitioning to rhizosphere respiration after a pulse labelling of maize assimilates

In a previous study, we examined the kinetics of radioactivity evolution from rhizosphere respiration after the pulse labelling of maize shoots with ¹⁴CO₂ (Nguyen et al., 1999). The specific activity of rhizosphere respiration demonstrated two peaks of ¹⁴CO₂ production. The first one occurred a few hours after the pulse of ¹⁴CO₂ and was followed by a second peak, which took place during the night following the labelling. In the present work, we demonstrate that the second phase of activity occurred in both sterile and non sterile plant–soil systems. This was inconsistent with the results obtained for wheat by Warembourg and Billès (1979) who observed the second peak solely in the case of non-sterile cultures. These authors suggested that this second phase of ¹⁴CO₂ production was related to microbial mineralisation of labelled complex compounds. Their synthesis and breakdown into smaller molecules delayed their utilisation by micro-organisms. However, in the present work, we also demonstrate that the second phase of activity was closely related to photoperiod. When plants were transferred from a 16 h to 20 h photoperiod, the second mineralisation of labelled rhizosphere compounds occurred sooner after the initiation of the dark period and it was strongly attenuated. Therefore, we suggest that the second phase of activity resulted from the utilisation by roots and by micro-organisms of stored ¹⁴C-compounds, which accumulated during the previous light period.     

Microbial methanol uptake in northeast Atlantic waters

Methanol is the predominant oxygenated volatile organic compound in the troposphere, where it can significantly influence the oxidising capacity of the atmosphere. However, we do not understand which processes control oceanic concentrations, and hence, whether the oceans are a source or a sink to the atmosphere. We report the first methanol loss rates in seawater by demonstrating that ¹⁴C-labelled methanol can be used to determine microbial uptake into particulate biomass, and oxidation to ¹⁴CO₂. We have found that methanol is used predominantly as a microbial energy source, but also demonstrated its use as a carbon source. We report biological methanol oxidation rates between 2.1 and 8.4 nmol l⁻¹ day⁻¹ in surface seawater of the northeast Atlantic. Kinetic experiments predict a V_max of up to 29 nmol l⁻¹ day⁻¹, with a high affinity K_m constant of 9.3 n in more productive coastal waters. We report surface concentrations of methanol in the western English channel of 97±8 n (n=4) between May and June 2010, and for the wider temperate North Atlantic waters of 70±13 n (n=6). The biological turnover time of methanol has been estimated between 7 and 33 days, although kinetic experiments suggest a 7-day turnover in more productive shelf waters. Methanol uptake rates into microbial particles significantly correlated with bacterial and phytoplankton parameters, suggesting that it could be used as a carbon source by some bacteria and possibly some mixotrophic eukaryotes. Our results provide the first methanol loss rates from seawater, which will improve the understanding of the global methanol budget.     

Paired-tower measurements of carbon and energy fluxes following disturbance in the boreal forest

Disturbances by fire and harvesting are thought to regulate the carbon balance of the Canadian boreal forest over scales of several decades. However, there are few direct measurements of carbon fluxes following disturbances to provide data needed to refine mathematical models. The eddy covariance technique was used with paired towers to measure fluxes simultaneously at disturbed and undisturbed sites over periods of about one week during the growing season in 1998 and 1999. Comparisons were conducted at three sites: a 1‐y‐old burned jackpine stand subjected to an intense crown fire at the International Crown Fire Modelling Experiment site near Fort Providence, North‐west Territories; a 1‐y‐old clearcut aspen area at the EMEND project near Peace River, Alberta; and a 10‐y‐old burned, mixed forest near Prince Albert National Park, Saskatchewan. Nearby mature forest stands of the same types were also measured as controls. The harvested site had lower net radiation (R_n), sensible (H) and latent (LE) heat fluxes, and greater ground heat fluxes (G) than the mature forest. Daytime CO₂ fluxes were much reduced, but night‐time CO₂ fluxes were identical to that of the mature aspen forest. It is hypothesized that the aspen roots remained alive following harvesting, and dominated soil respiration. The overall effect was that the harvested site was a carbon source of about 1.6 gC m^?2 day^?1, while the mature site was a sink of about ?3.8 gC m^?2 day^?1. The one‐year‐old burn had lower R_n, H and LE than the mature jackpine forest, and had a continuous CO₂ efflux of about 0.8 gC m^–2 day^?1 compared to the mature forest sink of ? 0.5 g C m^?2 day^?1. The carbon source was likely caused by decomposition of fire‐killed vegetation. The 10‐y‐old burned site had similar H, LE, and G to the mature mixed forest site. Although the diurnal amplitude of the CO₂ fluxes were slightly lower at the 10‐y‐old site, there was no significant difference between the daily integrals (? 1.3 gC m^?2 day^?1 at both sites). It appears that most of the change in carbon flux occurs within the first 10 years following disturbance, but more data are needed on other forest and disturbance types for the first 20 years following the disturbance event.     

Climatic variability,hydrologic anomaly,and methane emission can turn productive freshwater marshes into net carbon sources

Freshwater marshes are well‐known for their ecological functions in carbon sequestration, but complete carbon budgets that include both methane (CH₄) and lateral carbon fluxes for these ecosystems are rarely available. To the best of our knowledge, this is the first full carbon balance for a freshwater marsh where vertical gaseous [carbon dioxide (CO₂) and CH₄] and lateral hydrologic fluxes (dissolved and particulate organic carbon) have been simultaneously measured for multiple years (2011–2013). Carbon accumulation in the sediments suggested that the marsh was a long‐term carbon sink and accumulated ~96.9 ± 10.3 (±95% CI) g C m^?2 yr^?1 during the last ~50 years. However, abnormal climate conditions in the last 3 years turned the marsh to a source of carbon (42.7 ± 23.4 g C m^?2 yr^?1). Gross ecosystem production and ecosystem respiration were the two largest fluxes in the annual carbon budget. Yet, these two fluxes compensated each other to a large extent and led to the marsh being a CO₂ sink in 2011 (?78.8 ± 33.6 g C m^?2 yr^?1), near CO₂‐neutral in 2012 (29.7 ± 37.2 g C m^?2 yr^?1), and a CO₂ source in 2013 (92.9 ± 28.0 g C m^?2 yr^?1). The CH₄ emission was consistently high with a three‐year average of 50.8 ± 1.0 g C m^?2 yr^?1. Considerable hydrologic carbon flowed laterally both into and out of the marsh (108.3 ± 5.4 and 86.2 ± 10.5 g C m^?2 yr^?1, respectively). In total, hydrologic carbon fluxes contributed ~23 ± 13 g C m^?2 yr^?1 to the three‐year carbon budget. Our findings highlight the importance of lateral hydrologic inflows/outflows in wetland carbon budgets, especially in those characterized by a flow‐through hydrologic regime. In addition, different carbon fluxes responded unequally to climate variability/anomalies and, thus, the total carbon budgets may vary drastically among years.     

How strong is the current carbon sequestration of an Atlantic blanket bog?

Although northern peatlands cover only 3% of the land surface, their thick peat deposits contain an estimated one‐third of the world's soil organic carbon (SOC). Under a changing climate the potential of peatlands to continue sequestering carbon is unknown. This paper presents an analysis of 6 years of total carbon balance of an almost intact Atlantic blanket bog in Glencar, County Kerry, Ireland. The three components of the measured carbon balance were: the land‐atmosphere fluxes of carbon dioxide (CO₂) and methane (CH₄) and the flux of dissolved organic carbon (DOC) exported in a stream draining the peatland. The 6 years C balance was computed from 6 years (2003–2008) of measurements of meteorological and eddy‐covariance CO₂ fluxes, periodic chamber measurements of CH₄ fluxes over 3.5 years, and 2 years of continuous DOC flux measurements. Over the 6 years, the mean annual carbon was ?29.7±30.6 (±1 SD) g C m^?2 yr^?1 with its components as follows: carbon in CO₂ was a sink of ?47.8±30.0 g C m^?2 yr^?1; carbon in CH₄ was a source of 4.1±0.5 g C m^?2 yr^?1 and the carbon exported as stream DOC was a source of 14.0±1.6 g C m^?2 yr^?1. For 2 out of the 6 years, the site was a source of carbon with the sum of CH₄ and DOC flux exceeding the carbon sequestered as CO₂. The average C balance for the 6 years corresponds to an average annual growth rate of the peatland surface of 1.3 mm yr^?1.     

Plant rhizosphere influence on microbial C metabolism: the role of elevated CO2, N availability and root stoichiometry

Microbial decomposer C metabolism is considered a factor controlling soil C stability, a key regulator of global climate. The plant rhizosphere is now recognized as a crucial driver of soil C dynamics but specific mechanisms by which it can affect C processing are unclear. Climate change could affect microbial C metabolism via impacts on the plant rhizosphere. Using continuous ¹³C labelling under controlled conditions that allowed us to quantify SOM derived-C in all pools and fluxes, we evaluated the microbial metabolism of soil C in the rhizosphere of a C4 native grass exposed to elevated CO₂ and under variation in N concentrations in soil and in plant root C:N stoichiometry. Our results demonstrated that this plant can influence soil C metabolism and further, that elevated CO₂ conditions can alter this role by increasing microbial C efficiency as indicated by a reduction in soil-derived C respiration per unit of soil C-derived microbial biomass. Moreover, under elevated CO₂ increases in soil N, and notably, root tissue N concentration increased C efficiency, suggesting elevated CO₂ shifted the stoichiometric balance so N availability was a more critical factor regulating efficiency than under ambient conditions. The root C:N stoichiometry effect indicates that plant chemical traits such as root N concentration are able to influence the metabolism of soil C and that elevated CO₂ conditions can modulate this role. Increased efficiency in soil C use was associated with negative rhizosphere priming and we hypothesize that the widely observed phenomenon of rhizosphere priming may result, at least in part, from changes in the metabolic efficiency of microbial populations. Observed changes in the microbial community support that shifting microbial populations were a contributing factor to the observed metabolic responses. Our case study points at greater efficiency of the SOM-degrading populations in a high CO₂, high N world, potentially leading to greater C storage of microbially assimilated C in soil.     

Interactive effects of drought and N fertilization on the spatial distribution of methane assimilation in grassland soils

Soil methanotrophic bacteria constitute the only globally relevant biological sink for atmospheric methane (CH₄). Nitrogen (N) fertilizers as well as soil moisture regime affect the activity of these organisms, but the mechanisms involved are not well understood to date. In particular, virtually nothing is known about the spatial distribution of soil methanotrophs within soil structure and how this regulates CH₄ fluxes at the ecosystem scale. We studied the spatial distribution of CH₄ assimilation and its response to a factorial drought × N fertilizer treatment in a 3‐year experiment replicated in two grasslands differing in management intensity. Intact soil cores were labelled with ¹⁴CH₄ and methanotrophic activity mapped at a resolution of ～100 μm using an autoradiographic technique. Under drought, the main zone of CH₄ assimilation shifted down the soil profile. Ammonium nitrate (NH₄NO₃) and cattle urine reduced CH₄ assimilation in the top soil, but only when applied under drought, presumably because NH₄⁺ from fertilizers was not removed by plant uptake and nitrification under these conditions. Ecosystem‐level CH₄ fluxes measured in the field did show no or only very small inhibitory effects, suggesting that deeper soil layers fully compensated for the reduction in top soil CH₄ assimilation. Our results indicate that the ecosystem‐level CH₄ sink cannot be inferred from measurements of soil samples that do not reflect the spatial organization of soils (e.g. stratification of organisms, processes, and mechanisms). The autoradiographic technique we have developed is suited to study methanotrophic activity in a relevant spatial context and does not rely on the genetic identity of the soil bacterial communities involved, thus ideally complementing DNA‐based approaches.     

Contemporary carbon accumulation in a boreal oligotrophic minerogenic mire – a significant sink after accounting for all C-fluxes

Based on theories of mire development and responses to a changing climate, the current role of mires as a net carbon sink has been questioned. A rigorous evaluation of the current net C-exchange in mires requires measurements of all relevant fluxes. Estimates of annual total carbon budgets in mires are still very limited. Here, we present a full carbon budget over 2 years for a boreal minerogenic oligotrophic mire in northern Sweden (64°11′N, 19°33′E). Data on the following fluxes were collected: land–atmosphere CO₂ exchange (continuous Eddy covariance measurements) and CH₄ exchange (static chambers during the snow free period); TOC (total organic carbon) in precipitation; loss of TOC, dissolved inorganic carbon (DIC) and CH₄ through stream water runoff (continuous discharge measurements and regular C-concentration measurements). The mire constituted a net sink of 27±3.4 (±SD) g C m⁻² yr⁻¹ during 2004 and 20±3.4 g C m⁻² yr⁻¹ during 2005. This could be partitioned into an annual surface–atmosphere CO₂ net uptake of 55±1.9 g C m⁻² yr⁻¹ during 2004 and 48±1.6 g C m⁻² yr⁻¹ during 2005. The annual NEE was further separated into a net uptake season, with an uptake of 92 g C m⁻² yr⁻¹ during 2004 and 86 g C m⁻² yr⁻¹ during 2005, and a net loss season with a loss of 37 g C m⁻² yr⁻¹ during 2004 and 38 g C m⁻² yr⁻¹ during 2005. Of the annual net CO₂-C uptake, 37% and 31% was lost through runoff (with runoff TOC>DIC≫CH₄) and 16% and 29% through methane emission during 2004 and 2005, respectively. This mire is still a significant C-sink, with carbon accumulation rates comparable to the long-term Holocene C-accumulation, and higher than the C-accumulation during the late Holocene in the region.     

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.     

Feedback of carbon and nitrogen cycles enhances carbon sequestration in the terrestrial biosphere

The efforts to explain the ‘missing sink’ for anthropogenic carbon dioxide (CO₂) have included in recent years the role of nitrogen as an important constraint for biospheric carbon fluxes. We used the Nitrogen Carbon Interaction Model (NCIM) to investigate patterns of carbon and nitrogen storage in different compartments of the terrestrial biosphere as a consequence of a rising atmospheric CO₂ concentration, in combination with varying levels of nitrogen availability. This model has separate but closely coupled carbon and nitrogen cycles with a focus on soil processes and soil–plant interactions, including an active compartment of soil microorganisms decomposing litter residues and competing with plants for available nitrogen. Biological nitrogen fixation is represented as a function of vegetation nitrogen demand. The model was validated against several global datasets of soil and vegetation carbon and nitrogen pools. Five model experiments were carried out for the modeling periods 1860–2002 and 2002–2100. In these experiments we varied the nitrogen availability using different combinations of biological nitrogen fixation, denitrification, leaching of soluble nitrogen compounds with constant or rising atmospheric CO₂ concentrations. Oversupply with nitrogen, in an experiment with nitrogen fixation, but no nitrogen losses, together with constant atmospheric CO₂, led to some carbon sequestration in organismic pools, which was nearly compensated by losses of C from soil organic carbon pools. Rising atmospheric CO₂ always led to carbon sequestration in the biosphere. Considering an open nitrogen cycle including dynamic nitrogen fixation, and nitrogen losses from denitrification and leaching, the carbon sequestration in the biosphere is of a magnitude comparable to current observation based estimates of the ‘missing sink.’ A fertilization feedback between the carbon and nitrogen cycles occurred in this experiment, which was much stronger than the sum of separate influences of high nitrogen supply and rising atmospheric CO₂. The demand‐driven biological nitrogen fixation was mainly responsible for this result. For the modeling period 2002–2100, NCIM predicts continued carbon sequestration in the low range of previously published estimates, combined with a plausible rate of CO₂‐driven biological nitrogen fixation and substantial redistribution of nitrogen from soil to plant pools.     

Deeper Snow Enhances Winter Respiration from Both Plant-associated and Bulk Soil Carbon Pools in Birch Hummock Tundra

It has only recently become apparent that biological activity during winter in seasonally snow-covered ecosystems may exert a significant influence on biogeochemical cycling and ecosystem function. One-seventh of the global soil carbon pool is stored in the bulk soil component of arctic ecosystems. Consistent climate change predictions of substantial increases in winter air temperatures and snow depths for the Arctic indicate that this region may become a significant net annual source of CO₂ to the atmosphere if its bulk soil carbon is decomposed. We used snow fences to investigate the influence of a moderate increase in snow depth from approximately 0.3 m (ambient) to approximately 1 m on winter carbon dioxide fluxes from mesic birch hummock tundra in northern Canada. We differentiated fluxes derived from the bulk soil and plant-associated carbon pools using an experimental ‘weeding’ manipulation. Increased snow depth enhanced the wintertime carbon flux from both pools, strongly suggesting that respiration from each was sensitive to warmer soil temperatures. Furthermore, deepened snow resulted in cooler and relatively stable soil temperatures during the spring-thaw period, as well as delayed and fewer freeze–thaw cycles. The snow fence treatment increased mean total winter efflux from 27 to 43 g CO₂-C m⁻². Because total 2004 growing season net ecosystem exchange for this site is estimated at 29–37 g CO₂-C m⁻², our results strongly suggest that a moderate increase in snow depth can enhance winter respiration sufficiently to switch the ecosystem annual net carbon exchange from a sink to source, resulting in net CO₂ release to the atmosphere.     

Ecosystem fluxes of hydrogen in a mid‐latitude forest driven by soil microorganisms and plants

Molecular hydrogen (H₂) is an atmospheric trace gas with a large microbe‐mediated soil sink, yet cycling of this compound throughout ecosystems is poorly understood. Measurements of the sources and sinks of H₂ in various ecosystems are sparse, resulting in large uncertainties in the global H₂ budget. Constraining the H₂ cycle is critical to understanding its role in atmospheric chemistry and climate. We measured H₂ fluxes at high frequency in a temperate mixed deciduous forest for 15 months using a tower‐based flux‐gradient approach to determine both the soil‐atmosphere and the net ecosystem flux of H₂. We found that Harvard Forest is a net H₂ sink (?1.4 ± 1.1 kg H₂ ha^?1) with soils as the dominant H₂ sink (?2.0 ± 1.0 kg H₂ ha^?1) and aboveground canopy emissions as the dominant H₂ source (+0.6 ± 0.8 kg H₂ ha^?1). Aboveground emissions of H₂ were an unexpected and substantial component of the ecosystem H₂ flux, reducing net ecosystem uptake by 30% of that calculated from soil uptake alone. Soil uptake was highly seasonal (July maximum, February minimum), positively correlated with soil temperature and negatively correlated with environmental variables relevant to diffusion into soils (i.e., soil moisture, snow depth, snow density). Soil microbial H₂ uptake was correlated with rhizosphere respiration rates (r = 0.8, P < 0.001), and H₂ metabolism yielded up to 2% of the energy gleaned by microbes from carbon substrate respiration. Here, we elucidate key processes controlling the biosphere–atmosphere exchange of H₂ and raise new questions regarding the role of aboveground biomass as a source of atmospheric H₂ and mechanisms linking soil H₂ and carbon cycling. Results from this study should be incorporated into modeling efforts to predict the response of the H₂ soil sink to changes in anthropogenic H₂ emissions and shifting soil conditions with climate and land‐use change.