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Arbuscular mycorrhizal fungi (AMF) form mutualistic associations with roughly 70% of vascular plant species, supporting the nutrient acquisition of their host plants and deriving carbon in return. AMF and plant communities are linked to each other by host-specificity and the ecological selection of favorable nutrient and carbon trading strategies. Changing soil nutrient availabilities can affect both plant and AMF communities directly and also indirectly via the response of their partners. We aimed to elucidate the combined response of AMF (belowground) and plant (aboveground) community compositions to changing soil nutrient availabilities.We sampled soil and roots from a long-term nutrient deficiency experimental grassland in Admont (Styria, Austria). The grassland plots have been fertilized with different combinations of nitrogen (N), phosphorus (P), and potassium (K) over 70 years. Aboveground biomass cuts were removed three times each year, leading to long-term deficiencies of nutrients not replaced by fertilizers. Soil and root AMF community compositions were measured by DNA and RNA amplicon sequencing of the 18S rRNA gene. In addition, we assessed the plant community composition of the sampled roots by amplicon sequencing of the chloroplast rbcL (RuBisCo large subunit) gene region, and visually recorded the plant community composition on each investigated plot.Our results demonstrate that N and P deficiencies influenced soil AMF community composition, whereas K deficiency had a major impact on root AMF community composition. Interestingly, the plant community composition was affected by N and P, similar to the soil AMF community composition. Both, soil and root AMF community compositions were significantly correlated to plant community composition across all treatments, the correlation was however stronger for soil AMF communities (R2 = 0.55, p< 0.001). By using bipartite network analysis, we identified several fungus-plant pairs that responded consistently to treatments.Our results indicate that the response of grasslands to nutrient deficiencies is potentially driven by strong feedbacks between plant and belowground AMF community compositions. We here demonstrate that the known interactions between grassland plants and AMF - which are often investigated from a single plant or monoculture perspective - are major drivers of how diverse plant community compositions will respond to environmental change, such as fertilization. In conclusion, considering the ecology of the subsurface AMF communities may strongly benefit our understanding of plant communities in a future environment.

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13CO2 atmosphere. We analysed the short-term distribution of 13C and 15N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13C to root parts that received more 15N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13C and 15N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Trees allocate C from sources to sinks by way of a series of processes involving carbohydrate transport and utilization. Yet these dynamics are not well characterized in trees, and it is unclear how these dynamics will respond to a warmer world. Here, we conducted a warming and pulse-chase experiment on Eucalyptus parramattensis growing in a whole-tree chamber system to test whether warming impacts carbon allocation by increasing the speed of carbohydrate dynamics. We pulse-labelled large (6-m tall) trees with 13 C-CO2 to follow recently fixed C through different organs by using compound-specific isotope analysis of sugars. We then compared concentrations and mean residence times of individual sugars between ambient and warmed (+3°C) treatments. Trees dynamically allocated 13 C-labelled sugars throughout the aboveground-belowground continuum. We did not, however, find a significant treatment effect on C dynamics, as sugar concentrations and mean residence times were not altered by warming. From the canopy to the root system, 13 C enrichment of sugars decreased, and mean residence times increased, reflecting dilution and mixing of recent photoassimilates with older reserves along the transport pathway. Our results suggest that a locally endemic eucalypt was seemingly able to adjust its physiology to warming representative of future temperature predictions for Australia.

All plant species reach a low temperature range limit when either low temperature extremes exceed their freezing tolerance or when their metabolism becomes too restricted. In this study, we explore the ultimate thermal limit of plant tissue formation exemplified by a plant species that seemingly grows through snow. By a combination of studies in alpine snowbeds and under controlled environmental conditions, we demonstrate and quantify that the clonal herb Soldanella pusilla (Primulaceae) does indeed grow its entire flowering shoot at 0 °C. We show that plants resume growth under 2–3 m of snow in mid-winter, following an internal clock, with the remaining period under snow until snow melt (mostly in July) sufficient to produce a flowering shoot that is ready for pollination. When snow pack gets thin, the flowering shoot intercepts and re-radiates long-wave solar radiation, so that snow and ice gently melt around the fragile shoot and the flowers emerge without any mechanical interaction. We evidence bud preformation in the previous season and enormous non-structural carbohydrate reserves in tissues (mainly below ground) in the form of soluble sugars (largely stachyose) that would support basic metabolism for more than 2 entire years under snow. However, cell-wall formation at 0 °C appears to lack unknown strengthening factors, including lignification (assessed by confocal Raman spectroscopy imaging) that require between a few hours or a day of warmth after snow melt to complete tissue strengthening. Complemented with a suite of anatomical data, the work opens a window towards understanding low temperature limits of plant growth in general, with potential relevance for winter crops and trees at the natural climatic treeline.

Fertilization experiments provide insights into elemental imbalances in soil microbial communities and their consequences for soil nutrient cycling. By addition of selected nutrients, other nutrients become deficient and limiting for soil microorganisms as well as for plants. In this study we focused on microbial nitrogen (N) cycling in a long-term nutrient manipulation experiment. In many soils, the rate-limiting step in N cycling is depolymerization of high-molecular-weight nitrogen compounds (e.g., proteins) to oligomers (e.g., peptides) and monomers (e.g., amino acids) rather than the subsequent steps of mineralization (ammonification) and nitrification. The aim of our study was to determine whether nutrient deficiency directly or indirectly – via changes in plant carbon (C) inputs - affects soil microbial N processing.We collected soil samples from a fertilization experiment, established in 1946 on a hay meadow close to Admont (Styria, Austria). The field experiment consisted of a full factorial combination of inorganic N, P, and K fertilization and a control with no fertilizers. Furthermore, liming (Ca-addition) and organic fertilizer application treatments (solid manure and liquid slurry) were established. In the experiment, plant biomass is harvested three times per year, inducing strong nutrient limitation in plots that have not received nutrient additions (fully deficient or deficient in a single element). We determined gross rates of microbial protein depolymerization, N-mineralization and nitrification via isotope pool dilution assays with 15N-labeled amino acids, NH4+, and NO3-. We hypothesized that N deficiency (lack of N fertilization) would stimulate microbial N mining (depolymerization), and reduce subsequent N mineralization and nitrification. In contrast, we expected that organic fertilization would alleviate microbial C and N limitations, reducing N depolymerization rates and increasing mineralization and nitrification.Our results show that organically fertilized and limed soils have significantly lower gross protein depolymerization rates than plots receiving inorganic N. No significant differences were found comparing gross N-mineralization and gross nitrification rates across the different treatments. Given the higher rates of protein depolymerization in inorganically fertilized soils as compared to organically fertilized and limed soils, microbial N processes seem to be controlled by plant C input and/or soil pH rather than by direct soil nutrient availability. However, depolymerization of macromolecular N does not only supply N to the soil microbial community but also organic C. Thus, the reduced plant C input compared to fully fertilized soils may have caused microorganisms to increase their mining for a C-containing energy source, thereby increasing protein depolymerization rates. In summary, this study suggests that long term nutrient deficiency or nutrient imbalances may affect soil nutrient cycling indirectly by changing plant C inputs (via reduced primary production) and/or changing soil pH, rather than directly, by nutrient availability. This further indicates that soil microbial communities are rather C than nutrient limited.

Arbuscular mycorrhiza (AM) fungi are associated with almost all land plants and provide soil nutrients and other benefits to their plant hosts in exchange for photosynthetic products. While fertilization regimes in managed grasslands or agricultural systems are tailored for increasing plant biomass, their potential effects on AM fungi are rarely taken into account. Nutrient-driven changes in abundance and community composition of AM fungi, however, may feedback on ecosystem performance in the long term. Therefore, it is necessary to get a better understanding on how AM fungal communities respond to changes and imbalances in soil nutrient availabilities.Here, we evaluated how long-term nutrient deficiency of phosphorus (P), nitrogen (N) and potassium (K) affects the abundance and community composition of AM fungi in a mountainous grassland. In addition, we investigated how the responses of AM fungi to those deficiencies were modulated by liming and the type of fertilizer addition (inorganic versus organic).Our study was carried out on a long-term nutrient deficiency experimental grassland site in Admont (Styria, Austria), established in 1946. Different fertilization treatments were applied for more than 70 years in a randomized block design, including numerous combinations of inorganic (P, N, K with/without lime) and organic (solid manure and liquid slurry) fertilizers. The hay meadow at the site is cut three times per year and biomass is not returned to the system. Therefore, biomass and nutrients have been continuously removed for decades, leading to different types of soil nutrient deficiency. In this study, we collected both root and soil samples in July 2019 and quantified AM fungi and other microbial groups by measuring neutral fatty acid (NLFA) and phospholipid fatty acid (PLFA) biomarkers, respectively. Additionally, we applied DNA and RNA-based amplicon sequencing of the 18S rRNA gene to identify AM fungal community composition.Our data shows that deficiencies of one or more elements had a major impact on both AM fungal biomass and community composition. AM fungal biomass was higher in plots that received no fertilizers compared to inorganically fertilized plots, but lower in plots which were deficient only in certain single or multiple elements, specifically in plots fertilized with inorganic N only (i.e., deficient in P and K). Conversely, liming and organic fertilizer amendments increased AM fungal biomass compared to plots containing inorganic fertilizers without lime. Across all treatments, AM fungal biomass was positively correlated with pH and soil water content, and negatively with dissolved N compounds, indicating indirect effects via responses of other soil parameters to nutrient deficiency. Long-term nutrient deficiency also altered plant community composition, which may also have indirectly affected AM fungal communities.We conclude that long-term nutrient deficiency, and in particular the stoichiometry of available nutrients, strongly affects the abundance and community composition of AM fungi in grassland soil. This response may be linked to changes in plant community composition or soil chemistry both as a result and as a cause, emphasizing the complexity of feedbacks determining the response of grassland ecosystems to changing nutrient conditions.

Mycorrhizal fungi are an important partner of almost all land plants, who trade soil nutrients, such as Phosphorus or Nitrogen, for photosynthetic Carbon (C). Moreover, mycorrhizal fungi connect multiple plants with their mycelium in so called Common Mycorrhizal Networks (CMNs). CMNs formed by ectomycorrhizal (EM) fungi are an inherent part of boreal and temperate forests, often termed the ‘wood-wide web’. However, the role of these networks for plant belowground C allocation and distribution is not well known.Here, we examined how plant photosynthates are distributed within EM mycelium networks connecting pairs of young beech trees, addressing the following questions: (1) Is the total belowground C allocation of plant photosynthates influenced by the size of the mycorrhizal network and its access to resources? (2) Is the belowground C distribution within a CMN altered if trees have unequal access to C from photosynthesis? (3) Do CMNs amplify or alleviate competition for nutrients between connected trees?We planted young beech trees in pots in a special two-plant box set-up which allows to control the establishment of mycorrhizal networks between them. For this, two plant pots, penetrable by fungal hyphae but not by roots, were placed inside of plastic boxes and the interstitial space was filled with quartz sand. In addition, a hyphal-exclusive N source consisting of 15N labeled peat (‘peat bag’), was buried within each plant pot. Two treatments were applied in a fully factorial design: 1) Allowing/preventing the establishment of a CMN between the pots (some pots were turned around at a regular interval to prevent the establishment of CMNs) and 2) inequality of access to photoassimilated C (in part of the boxes one of the two plants was shaded). In a 13C-CO2 labeling approach, we traced 13C assimilated by one plant of each tree pair into belowground pools of both plants by isotope ratio mass spectrometry (EA-IRMS) and 13C phospholipid fatty acid (PLFAs) analysis (GC-IRMS). At the same time, we investigated plant uptake of 15N via mycorrhiza by EA-IRMS.Our results demonstrate that plants relied mostly on their fungal partners to acquire nutrients (63% of plant N was derived from mycorrhiza-exclusive peat bags), and also directed the majority of the C allocated belowground to their mycorrhizal partners. The presence of a larger mycorrhizal network connecting to another plant and an additional N source almost doubled photosynthetic CO2 assimilation and belowground C allocation by plants. Fungi translocated carbon via hyphal linkages preferentially into mycorrhiza-exclusive nutrient patches, even when they were located within the realm of a neighboring plant and this necessitates to cross a nutrient-poor zone of sand. Shading did not affect the belowground distribution of C.We conclude that belowground ectomycorrhizal networks represent a significant sink strength for plant photosynthates and may thus be a major driver of C sequestration in beech forest soils. The belowground distribution of C via fungal networks is mainly related to the distribution of nutrient-rich patches in the soil and less to differences in the photosynthetic capacity of the host plants.

Non-structural carbohydrates (NSCs), the stored products of photosynthesis, building blocks for growth and fuel for respiration, are central to plant metabolism, but their measurement is challenging. Differences in methods and procedures among laboratories can cause results to vary widely, limiting our ability to integrate and generalize patterns in plant carbon balance among studies. A recent assessment found that NSC concentrations measured for a common set of samples can vary by an order of magnitude, but sources for this variability were unclear. We measured a common set of nine plant material types, and two synthetic samples with known NSC concentrations, using a common protocol for sugar extraction and starch digestion, and three different sugar quantification methods (ion chromatography, enzyme, acid) in six laboratories. We also tested how sample handling, extraction solvent and centralizing parts of the procedure in one laboratory affected results. Non-structural carbohydrate concentrations measured for synthetic samples were within about 11.5% of known values for all three methods. However, differences among quantification methods were the largest source of variation in NSC measurements for natural plant samples because the three methods quantify different NSCs. The enzyme method quantified only glucose, fructose and sucrose, with ion chromatography we additionally quantified galactose, while the acid method quantified a large range of mono- and oligosaccharides. For some natural samples, sugars quantified with the acid method were two to five times higher than with other methods, demonstrating that trees allocate carbon to a range of sugar molecules. Sample handling had little effect on measurements, while ethanol sugar extraction improved accuracy over water extraction. Our results demonstrate that reasonable accuracy of NSC measurements can be achieved when different methods are used, as long as protocols are robust and standardized. Thus, we provide detailed protocols for the extraction, digestion and quantification of NSCs in plant samples, which should improve the comparability of NSC measurements among laboratories.