▪ Abstract The use of stable isotope techniques in plant ecological research has grown steadily during the past two decades. This trend will continue as investigators realize that stable isotopes can serve as valuable nonradioactive tracers and nondestructive integrators of how plants today and in the past have interacted with and responded to their abiotic and biotic environments. At the center of nearly all plant ecological research which has made use of stable isotope methods are the notions of interactions and the resources that mediate or influence them. Our review, therefore, highlights recent advances in plant ecology that have embraced these notions, particularly at different spatial and temporal scales. Specifically, we review how isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen our understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies. Where possible we also...

Stable Isotopes in Plant Ecology

Equilibrium and kinetic isotope fractionations during incomplete reactions result in minute differences in the ratio between the two stable N isotopes, 15N and 14N, in various N pools. In ecosystems such variations (usually expressed in per mil [δ15N] deviations from the standard atmospheric N2) depend on isotopic signatures of inputs and outputs, the input–output balance, N transformations and their specific isotope effects, and compartmentation of N within the system. Products along a sequence of reactions, e.g. the N mineralization–N uptake pathway, should, if fractionation factors were equal for the different reactions, become progressively depleted. However, fractionation factors vary. For example, because nitrification discriminates against 15N in the substrate more than does N mineralization, NH4+ can become isotopically heavier than the organic N from which it is derived.Levels of isotopic enrichment depend dynamically on the stoichiometry of reactions, as well as on specific abiotic and biotic conditions. Thus, the δ15N of a specific N pool is not a constant, and δ15N of a N compound added to the system is not a conservative, unchanging tracer. This fact, together with analytical problems of measuring δ15N in small and dynamic pools of N in the soil–plant system, and the complexity of the N cycle itself (for instance the abundance of reversible reactions), limit the possibilities of making inferences based on observations of 15N abundance in one or a few pools of N in a system. Nevertheless, measurements of δ15N might offer the advantage of giving insights into the N cycle without disturbing the system by adding 15N tracer.Such attempts require, however, that the complex factors affecting δ15N in plants be taken into account, viz. (i) the source(s) of N (soil, precipitation, NOx, NH3, N2-fixation), (ii) the depth(s) in soil from which N is taken up, (iii) the form(s) of soil-N used (organic N, NH4+, NO3−), (iv) influences of mycorrhizal symbioses and fractionations during and after N uptake by plants, and (v) interactions between these factors and plant phenology. Because of this complexity, data on δ15N can only be used alone when certain requirements are met, e.g. when a clearly discrete N source in terms of amount and isotopic signature is studied. For example, it is recommended that N in non-N2-fixing species should differ more than 5‰ from N derived by N2-fixation, and that several non-N2-fixing references are used, when data on δ15N are used to estimate N2-fixation in poorly described ecosystems.As well as giving information on N source effects, δ15N can give insights into N cycle rates. For example, high levels of N deposition onto previously N-limited systems leads to increased nitrification, which produces 15N-enriched NH4+ and 15N-depleted NO3−. As many forest plants prefer NH4+ they become enriched in 15N in such circumstances. This change in plant δ15N will subsequently also occur in the soil surface horizon after litter-fall, and might be a useful indicator of N saturation, especially since there is usually an increase in δ15N with depth in soils of N-limited forests.Generally, interpretation of 15N measurements requires additional independent data and modelling, and benefits from a controlled experimental setting. Modelling will be greatly assisted by the development of methods to measure the δ15N of small dynamic pools of N in soils. Direct comparisons with parallel low tracer level 15N studies will be necessary to further develop the interpretation of variations in δ15N in soil–plant systems. Another promising approach is to study ratios of 15N[ratio ]14N together with other pairs of stable isotopes, e.g. 13C[ratio ]12C or 18O[ratio ]16O, in the same ion or molecules. This approach can help to tackle the challenge of distinguishing isotopic source effects from fractionations within the system studied.

Tansley Review No. 95 15N natural abundance in soil-plant systems

Boreal forest soils function as a terrestrial net sink in the global carbon cycle. The prevailing dogma has focused on aboveground plant litter as a principal source of soil organic matter. Using C-14 bomb-carbon modeling, we show that 50 to 70% of stored carbon in a chronosequence of boreal forested islands derives from roots and root-associated microorganisms. Fungal biomarkers indicate impaired degradation and preservation of fungal residues in late successional forests. Furthermore, 454 pyrosequencing of molecular barcodes, in conjunction with stable isotope analyses, highlights root-associated fungi as important regulators of ecosystem carbon dynamics. Our results suggest an alternative mechanism for the accumulation of organic matter in boreal forests during succession in the long-term absence of disturbance.

http://science.sciencemag.org/content/sci/339/6127/1615.full.pdf

Roots and associated fungi drive long-term carbon sequestration in boreal forest.

Our understanding of how saprotrophic and mycorrhizal fungi interact to re-circulate carbon and nutrients from plant litter and soil organic matter is limited by poor understanding of their spatiotemporal dynamics. In order to investigate how different functional groups of fungi contribute to carbon and nitrogen cycling at different stages of decomposition, we studied changes in fungal community composition along vertical profiles through a Pinus sylvestris forest soil. We combined molecular identification methods with 14C dating of the organic matter, analyses of carbon:nitrogen (C:N) ratios and 15N natural abundance measurements. Saprotrophic fungi were primarily confined to relatively recently (< 4 yr) shed litter components on the surface of the forest floor, where organic carbon was mineralized while nitrogen was retained. Mycorrhizal fungi dominated in the underlying, more decomposed litter and humus, where they apparently mobilized N and made it available to their host plants. Our observations show that the degrading and nutrient-mobilizing components of the fungal community are spatially separated. This has important implications for biogeochemical studies of boreal forest ecosystems.

/pdf/spatial-separation-of-litter-decomposition-and-mycorrhizal-57wp06kh9t.pdf

Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest

https://www.cell.com/trends/plant-science/pdf/S1360-1385(01)01889-1.pdf

Physiological mechanisms influencing plant nitrogen isotope composition.

15N natural abundances of soil total N, roots and mycorrhizas were studied in surface soil profiles in coniferous and broadleaved forests along a transect from central to northern Europe. Under conditions of N limitation in Sweden, there was an increase in δ15N of soil total N of up to 9% from the uppermost horizon of the organic mor layer down to the upper 0-5 cm of the mineral soil. The δ15N of roots was only slightly lower than that of soil total N in the upper organic horizon, but further down roots were up to 5% depleted under such conditions. In experimentally N-enriched forest in Sweden, i.e. in plots which have received an average of c. 100 kg N ha-1 year-1 for 20 years and which retain less than 50% of this added N in the stand and the soil down to 20 cm depth, and in some forests in central Europe, the increase in δ15N with depth in soil total N was smaller. An increase in δ15N of the surface soil was even observed on experimentally N-enriched plots, although other data suggest that the N fertilizer added was depleted in15N. In such cases roots could be enriched in15N relative to soil total N, suggesting that labelling of the surface soil is via the pathway: - available pools of N-plant N-litter N. Under N-limiting conditions roots of different species sampled from the same soil horizon showed similar δ15N. By contrast, in experimentally N-enriched forest δ15N of roots increased in the sequence: ericaceous dwarf shrubs<Scots pine<grass, suggesting increasing use of inorganic N along the sequence. Complementary studies at the major transect sites had shown that 90-99% of fine tree roots had ectomycorrhizas (ECMs). ECMs were 2% more enriched than corresponding non-mycorrhizal fine roots. Fungal sheaths stripped off ECMs were 2.4-6.4 enriched relative to the remaining root core. It is suggested that a flux of N through ECMs to aboveground parts in N-limited forests would leave 15N enriched compounds in fungal material, which could contribute to explain the observed δ15N profiles if fungal material is enriched, because it is a precursor of stable organic matter and recalcitrant N. This could act in addition to the previous explanation of the isotopically lighter soil surface in forests: plant uptake of 15N-depleted N and its redeposition onto the soil surface by litter-fall.

15N abundance of surface soils, roots and mycorrhizas in profiles of European forest soils.

A direct correlation was found between fractional losses of added N and the change in δ15N‰ during 19 years in an experiment with annual additions of N at three rates to a Scots pine (Pinus sylvestris L.) forest in northern Sweden. This confirms that processes leading to losses of N discriminate against15N, and opens possibilities to conduct retrospective studies of the N balance in forests.

15N Abundance of forests is correlated with losses of nitrogen

Summary
•Trees reduce their carbon (C) allocation to roots and mycorrhizal fungi in response to high nitrogen (N) additions, which should reduce the N retention capacity of forests. The time needed for recovery of mycorrhizas after termination of N loading remains unknown.
•Here, we report the long-term impact of N loading and the recovery of ectomycorrhiza after high N loading on a Pinus sylvestris forest. We analysed the N% and abundance of the stable isotope 15N in tree needles and soil, soil microbial fatty acid biomarkers and fungal DNA.
•Needles in N-loaded plots became enriched in 15N, reflecting decreased N retention by mycorrhizal fungi and isotopic discrimination against 15N during loss of N. Meanwhile, needles in N-limited (control) plots became depleted in 15N, reflecting high retention of 15N by mycorrhizal fungi. N loading was terminated after 20 yr. The δ15N and N% of the needles decreased 6 yr after N loading had been terminated, and approached values in control plots after 15 yr. This decrease, and the larger contributions compared with N-loaded plots of a fungal fatty acid biomarker and ectomycorrhizal sequences, suggest recovery of ectomycorrhiza.
•High N loading rapidly decreased the functional role of ectomycorrhiza in the forest N cycle, but significant recovery occurred within 6–15 yr after termination of N loading.

Recovery of ectomycorrhiza after 'nitrogen saturation' of a conifer forest.

15N abundances of soils and a grass species (Deschampsia flexuosa (L.) Trin.) were analysed in a forest fertilization experiment 10 years after the last fertilization. Nitrogen had been given as urea, at seven doses, ranging from 0 to 2400 kg N ha-1. Previously, we have shown that plants in systems experiencing large losses of N become enriched with 15N. This was explained by the fact that processes leading to loss of N, e.g. ammonia volatilization, nitrification followed by leaching or denitrification and denitrification itself, tend to fractionate against 15N. In this experiment, 15N abundance increased with dose of N applied in both grass and soil total-N, but more so in the grass. This was interpreted to be due to the grass sampling small but active pools of N subject to losses. In contrast, soil total-N largely consists of inactive N that does not immediately exchange with pools of N from which fractionating losses occur. Hence, soil total-N shows a large pretreatment 15N memory effect, and is, therefore, and integrator of the long-term N balance. When short-term changes (years, decades) in N balances are monitored using variations in 15N abundance, plants are more suitable indicators of such change than is soil total-N.

15N abundance of soils and plants along an experimentally induced forest nitrogen supply gradient.

Addition of N to an initially N-limited forest increases foliage biomass, demand for water and the probability of water stress. Effects of water and N on tree growth are thus compounded. The 13C abundance of plant tissues is directly correlated with water use efficiency (WUE), and could be used to disentangle the effect of water alone on carbon fixation. However, the 13C abundance may also be directly influenced by changes in rates of photosynthesis related to variations in N status, and by variations in N metabolism via non-RuBisCo carboxylations, and indirectly by effects of N source on WUE. We studied the 13C abundance of current needles from top whorls in two long-term fertilization experiments, one in Norway spruce (Picea abies Karst.) and one in Scots pine (Pinus sylvestris L.). As predicted, N fertilization increased foliage biomass and δ (‰). In the experiment with spruce this effect on 13C abundance was correlated with volume production and foliage biomass in a dry year, but was not seen in a wet year after 19 years of continuous annual N fertilization, which rules out the possible influences of N metabolism and changes in rates of photosynthesis. In the experiment with pine, which was at a drier site, needles from N-fertilized plots had a higher 13C abundance in three dry years, but not significantly so in a wet year. We suggest that effects of N source (NH4+ or NO3−) on 13C abundance are unlikely to be important under these experimental conditions. The balance between demand and supply of water should thus be the major determinant of the 13C abundance of current needles on top whorls. This opens possibilities to conduct retrospective studies of the role of water supply in fertilization experiments.

Christian Johannisson

Papers

15N abundance of surface soils, roots and mycorrhizas in profiles of European forest soils.

15N Abundance of forests is correlated with losses of nitrogen

Recovery of ectomycorrhiza after 'nitrogen saturation' of a conifer forest.

15N abundance of soils and plants along an experimentally induced forest nitrogen supply gradient.

Studies of 13C in the foliage reveal interactions between nutrients and water in forest fertilization experiments