Torgny Näsholm

Boreal forest plants take up organic nitrogen

Plant growth in the boreal forest, the largest terrestrial biome, is generally limited by the availability of nitrogen. The presumed cause of this limitation is slow mineralization of soil organic nitrogen,. Here we demonstrate, to our knowledge for the first time, the uptake of organic nitrogen in the field by the trees Pinus sylvestris and Picea abies, the dwarf shrub Vaccinium myrtillus and the grass Deschampsia flexuosa. These results show that these plants, irrespective of their different types of root–fungal associations (mycorrhiza), bypass nitrogen mineralization. A trace of the amino acid glycine, labelled with the stable isotopes 13C and 15N, was injected into the organic (mor) layer of an old successional boreal coniferous forest. Ratios of 13C:15N in the roots showed that at least 91, 64 and 42% of the nitrogen from the absorbed glycine was taken up in intact glycine by the dwarf shrub, the grass and the trees, respectively. Rates of glycine uptake were similar to those of 15N-ammonium. Our data indicate that organic nitrogen is important for these different plants, even when they are competing with each other and with non-symbiotic microorganisms. This has major implications for our understanding of the effects of nitrogen deposition, global warming and intensified forestry.

The unexpected versatility of plants: organic nitrogen use and availability in terrestrial ecosystems.

The recently recognized importance of organic nitrogen (ON), particularly amino acids, to plant nutrition in many types of agricultural and natural ecosystems has raised questions about plant-microbe interactions, N availability in soils, and the ecological implications of ON use by plants in the light of climate change and N pollution. In this review we synthesize the recent work on availability and plant uptake of amino acids with classic work on ON in soils. We also discuss recent work on the use of natural abundance levels of 15N to infer N sources for plants. Reliance on ON is widespread among plants from many ecosystems. Authors have reached this conclusion based on laboratory studies of amino acid uptake by plants in pure culture, amino acid concentrations in soils, plant uptake of isotopically labeled amino acids in the field and in plant-soil microcosms, and from plant natural abundance values of 15N. The supply of amino acids to plants is determined mainly by the action of soil proteolytic enzymes, interactions between amino acids and the soil matrix, and competition between plants and microbes. Plants generally compete for a minor fraction of the total amino acid flux, but in some cases this forms a significant N resource, especially in ecosystems where microbial biomass undergoes large seasonal fluctuations and contributes labile ON to the soil. A quantitative understanding of ON use by plants is confounded by incomplete data on partitioning of ON between plants, mycorrhizal fungi, and competing soil microbes. Further research is needed to predict the ecological implications of ON use by plants given the influence of climatic change and N pollution.

UPTAKE OF ORGANIC NITROGEN IN THE FIELD BY FOUR AGRICULTURALLY IMPORTANT PLANT SPECIES

Uptake of glycine was studied in four plants commonly used in grasslands in northern Europe (Phleum pratense, Trifolium hybridum, T. pratense, and Ranunculus acris) and compared to uptake of ammonium and nitrate. The experiment was conducted in the field, but with plants transferred to pots with soil 8–10 d before the start of the experiment. Plant uptake of U-13C215N glycine, 15NH4+, and 15NO3− was studied by injecting dilute (1 mmol/L) solutions of respectively labeled N source into the pots and harvesting plants 21 h later. Measurements of 13C and 15N in roots showed that, in all plants, part of the glycine N was taken up in the form of intact amino acid. Hence, regressions of plots of excess 13C against excess 15N showed that a minimum of 19–23% of the glycine-derived N was taken up as intact amino acid; possible losses of labeled C atoms of glycine during its metabolism in the plants implies that these estimates are conservative. Uptake of the different N sources was similar in the two Trifolium spec...

Nitrogen deposition and the biodiversity of boreal forests: implications for the nitrogen critical load.

Abstract The critical load concept is used to establish the deposition levels which ecosystems can tolerate without significant harmful effects. Here we summarize work within the Swedish research program Abatement Strategies for Transboundary Air Pollution (ASTA) assessing the critical load of N for boreal forests. Results from both field experiments in an area with low background N deposition in northern Sweden, and from a large-scale monitoring study, show that important vegetational changes start to take place when adding low N doses and that recovery of the vegetation after ceasing N input is a very slow process. The data presented indicate that changes in key ecosystem components occur even at a lower rate of N input than the present recommended empirical critical load for boreal forest understorey vegetation of 10–15 kg N ha−1 yr−1. Based on the data presented, we suggest that the critical load should be lowered to 6 kg N ha−1 yr−1.

A conditional marker gene allowing both positive and negative selection in plants

Selectable markers enable transgenic plants or cells to be identified after transformation. They can be divided into positive and negative markers conferring a selective advantage or disadvantage, respectively. We present a marker gene, dao1, encoding D-amino acid oxidase (DAAO, EC 1.4.3.3) that can be used for either positive or negative selection, depending on the substrate. DAAO catalyzes the oxidative deamination of a range of D-amino acids. Selection is based on differences in the toxicity of different D-amino acids and their metabolites to plants. Thus, D-alanine and D-serine are toxic to plants, but are metabolized by DAAO into nontoxic products, whereas D-isoleucine and D-valine have low toxicity, but are metabolized by DAAO into the toxic keto acids 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively. Hence, both positive and negative selection is possible with the same marker gene. The marker has been successfully established in Arabidopsis thaliana, and proven to be versatile, rapidly yielding unambiguous results, and allowing selection immediately after germination.

Comprehensive Screening of Arabidopsis Mutants Suggests the Lysine Histidine Transporter 1 to Be Involved in Plant Uptake of Amino Acids

Plant nitrogen (N) uptake is a key process in the global N cycle and is usually considered a “bottleneck” for biomass production in land ecosystems. Earlier, mineral N was considered the only form available to plants. Recent studies have questioned this dogma and shown that plants may access organic N sources such as amino acids. The actual mechanism enabling plants to access amino acid N is still unknown. However, a recent study suggested the Lysine Histidine Transporter 1 (LHT1) to be involved in root amino acid uptake. In this study, we isolated mutants defective in root amino acid uptake by screening Arabidopsis (Arabidopsis thaliana) seeds from ethyl methanesulfonate-treated plants and seeds from amino acid transporter T-DNA knockout mutants for resistance against the toxic d-enantiomer of alanine (Ala). Both ethyl methanesulfonate and T-DNA knockout plants identified as d-Ala resistant were found to be mutated in the LHT1 gene. LHT1 mutants displayed impaired capacity for uptake of a range of amino acids from solutions, displayed impaired growth when N was supplied in organic forms, and acquired substantially lower amounts of amino acids than wild-type plants from solid growth media. LHT1 mutants grown on mineral N did not display a phenotype until at the stage of flowering, when premature senescence of old leaf pairs occurred, suggesting that LHT1 may fulfill an important function at this developmental stage. Based on the broad and unbiased screening of mutants resistant to d-Ala, we suggest that LHT1 is an important mediator of root uptake of amino acids. This provides a molecular background for plant acquisition of organic N from the soil.

Are ectomycorrhizal fungi alleviating or aggravating nitrogen limitation of tree growth in boreal forests

Symbioses between plant roots and mycorrhizal fungi are thought to enhance plant uptake of nutrients through a favourable exchange for photosynthates. Ectomycorrhizal fungi are considered to play this vital role for trees in nitrogen (N)-limited boreal forests. We followed symbiotic carbon (C)-N exchange in a large-scale boreal pine forest experiment by tracing (13) CO(2) absorbed through tree photosynthesis and (15) N injected into a soil layer in which ectomycorrhizal fungi dominate the microbial community. We detected little (15) N in tree canopies, but high levels in soil microbes and in mycorrhizal root tips, illustrating effective soil N immobilization, especially in late summer, when tree belowground C allocation was high. Additions of N fertilizer to the soil before labelling shifted the incorporation of (15) N from soil microbes and root tips to tree foliage. These results were tested in a model for C-N exchange between trees and mycorrhizal fungi, suggesting that ectomycorrhizal fungi transfer small fractions of absorbed N to trees under N-limited conditions, but larger fractions if more N is available. We suggest that greater allocation of C from trees to ectomycorrhizal fungi increases N retention in soil mycelium, driving boreal forests towards more severe N limitation at low N supply.

The below‐ground perspective of forest plants: soil provides mainly organic nitrogen for plants and mycorrhizal fungi

• Nitrogen (N) availability has a major impact on a wide range of biogeochemical processes in terrestrial ecosystems. Changes in N availability modify the capacity of plants to sequester carbon (C), but despite the crucial importance for our understanding of terrestrial ecosystems, the relative contribution of different N forms to plant N nutrition in the field is not known. Until now, reliably assessing the highly dynamic pool of plant-available N in soil microsites was virtually impossible, because of the lack of adequate sampling techniques. • For the first time we have applied a novel microdialysis technique for disturbance-free monitoring of diffusive fluxes of inorganic and organic N in 15 contrasting boreal forest soils in situ. • We found that amino acids accounted for 80% of the soil N supply, while ammonium and nitrate contributed only 10% each. In contrast to common soil extractions, microdialysis revealed that the majority of amino acids are available for plant and mycorrhizal uptake. • Our results suggest that the N supply of boreal forest soils is dominated by organic N as a major component of plant-available N and thus as a regulator of growth and C sequestration.

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.

Recovery of ectomycorrhiza after ‘nitrogen saturation’ of a conifer forest

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 (15) N in tree needles and soil, soil microbial fatty acid biomarkers and fungal DNA. Needles in N-loaded plots became enriched in (15) N, reflecting decreased N retention by mycorrhizal fungi and isotopic discrimination against (15) N during loss of N. Meanwhile, needles in N-limited (control) plots became depleted in (15) N, reflecting high retention of (15) N by mycorrhizal fungi. N loading was terminated after 20yr. The δ(15) N and N% of the needles decreased 6yr after N loading had been terminated, and approached values in control plots after 15yr. 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 6-15yr after termination of N loading.