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.     

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.     

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.     

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.