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Increasing plants photosynthetic efficienc y is a major challenge that must be addressed in order to cover the food demands of the growing population in the changing climate. Photosynthes i s is greatly limited at the initial carboxylation reaction, where CO2 is converted to the organic acid 3-PGA, catalyzed by the RuBisCO enzyme. RuBisCO has poor affinity for CO2, but also the CO2 concentration at the RuBisCO site is limited by the diffusion of atmospheric CO2 through the various leaf compartments to the reaction site. Beyond genetic engineer-ing, nanotechnology can offer a materials-based approach for enhancing photosynthesis, and yet, it has mostly been explored for the light-dependent reactions. In this work, we developed polyethyleneimine-based nanoparticl e s for enhancing the carbox-ylation reaction. We demonstrate that the nanoparticles can capture CO2 in the form of bicarbonate and increase the CO2 that reacts with the RuBisCO enzyme, enhancing the 3-PGA production in in vitro assays by 20%. The nanoparticles can be introduced to the plant via leaf infiltration and, because of the functionalization with chitosan oligomers, they do not induce any toxic effect to the plant. In the leaves, the nanoparticles localize in the apoplastic space but also spontaneously reach the chloroplasts where photosynthetic activity takes place. Their CO2 loading-dependent fluorescence verifies that, in vivo, they maintain their abi l i t y to capture CO2 and can be therefore reloaded with atmospheric CO2 while in planta. Our results contribute to the development of a nanomaterials-based CO2-concentrating mechanism in plants t h a t can potentially increase photosynthetic efficiency and overall plants CO2 storage. Funding Agencies|European Union [800926, 101042148]; Swedish Research Council [VR-2017-04910]; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University [2009-00971]; MCIN/AEI [Ayuda RYC2021-031668-I]

Here we present a novel model supplementing existing theories of soil organic matter (SOM) decomposition, based on evolutionary-ecological principles rather than chemical or physical limitations to decomposition. We argue that decomposition of some substrates, in particular nitrogen-rich non-hydrolyzable matter (NHLS), may be constrained by spatial competition from opportunists (Bonner et al., 2022). Our model is based on two linked hypotheses: (1) From an evolutionary point of view, microbes should optimise their enzyme production to maximise the net fitness gain (F), and they should only decompose NHLS if the uptake of decomposition products (S) brings a net fitness gain (F > 0) in terms of growth minus costs of enzyme production. (2) F strongly depends on the fraction of decomposition products absorbed by the decomposer, i.e. the return on enzyme investment, which depends on the distance to the substrate and the competition from opportunistic bacteria. A minimum ‘safe’ distance for oxidative decomposition is included, based on the idea that cost of oxidative stress to the decomposer will surpass potential gain from decomposition when the activity is too close. Although the model predictions have not been tested directly against observations, they provide proof-of-concept that substrate can be spared decomposition and accumulate even when it is physically and chemically accessible. Due to the spatial competition effect, it is not profitable for either bacteria or decomposer fungi to decompose NHLS under certain conditions. Our framework can help explain a variety of SOM dynamics, including priming and the suppression of decomposition by nitrogen addition. ReferenceBonner MTL, Franklin O, Hasegawa S, Näsholm T. 2022. Those who can don't want to, and those who want to can't: An eco-evolutionary mechanism of soil carbon persistence. Soil Biology and Biochemistry 174: 108813.

Seminal scientific papers positing that mycorrhizal fungal networks can distribute carbon (C) among plants have stimulated a popular narrative that overstory trees, or 'mother trees', support the growth of seedlings in this way. This narrative has far-reaching implications for our understanding of forest ecology and has been controversial in the scientific community. We review the current understanding of ectomycorrhizal C metabolism and observations on forest regeneration that make the mother tree narrative debatable. We then re-examine data and conclusions from publications that underlie the mother tree hypothesis. Isotopic labeling methods are uniquely suited for studying element fluxes through ecosystems, but the complexity of mycorrhizal symbiosis, low detection limits, and small carbon discrimination in biological processes can cause researchers to make important inferences based on miniscule shifts in isotopic abundance, which can be misleading. We conclude that evidence of a significant net C transfer via common mycorrhizal networks that benefits the recipients is still lacking. Furthermore, a role for fungi as a C pipeline between trees is difficult to reconcile with any adaptive advantages for the fungi. Finally, the hypothesis is neither supported by boreal forest regeneration patterns nor consistent with the understanding of physiological mechanisms controlling mycorrhizal symbiosis.

The interaction between plants and plant pathogens can have significant effects on ecosystem performance. For their growth and development, both bionts rely on amino acids. While amino acids are key transport forms of nitrogen and can be directly absorbed from the soil through specific root amino acid transporters, various pathogenic microbes can invade plant tissues to feed on different plant amino acid pools. In parallel, plants may initiate an immune response program to restrict this invasion, employing various amino acid transporters to modify the amino acid pool at the site of pathogen attack. The interaction between pathogens and plants is sophisticated and responses are dynamic. Both avail themselves of multiple tools to increase their chance of survival. In this review, we highlight the role of amino acid transporters during pathogen infection. Having control over the expression of those transporters can be decisive for the fate of both bionts but the underlying mechanism that regulates the expression of amino acid transporters is not understood to date. We provide an overview of the regulation of a variety of amino acid transporters, depending on interaction with biotrophic, hemibiotrophic or necrotrophic pathogens. In addition, we aim to highlight the interplay of different physiological processes on amino acid transporter regulation during pathogen attack and chose the LYSINE HISTIDINE TRANSPORTER1 (LHT1) as an example.

There is evidence that carbon fluxes and stocks decrease with increasing latitude in boreal forests, suggesting areduction in carbon use efficiency. While vegetation and soi! carbon dynamics have been widely studied, the empirical finding that ectomycorrhizal fungi (ECM) become more abundant towards the north has not been quantitatively linked to carbon use efficiency. We formulated a conceptual model of combined fine-root and ECM carbon use efficiency (CUE) as NPP /GPP (net primary production/gross primary production). For this, we included the mycorrhiza as gains in plant NPP but considered the extramatrical hyphae as well as exudates as losses. We quantified the carbon processes across a latitudinal gradient using published eco-physiological and morphological measurements from boreal coniferous forests. !n parallel, we developed two CUE models using large-scale empirical measurements amended with established models. Ali models predicted similar latitudinal trends in vegetation CUE and net ecosystem production (NEP). CUE in the ECM model declined on average by 0.1 from latitude 60 to 70 with overall mean 0.390 ± 0.037. NEP declined by 200 g m-2 yr-1 with mean 171 ± 79.4 g m-2 yr-1• ECM had no significant effect on predicted soi! carbon. Our findings suggest that ECM can use a significant proportion of the carbon assimilated by vegetation and hence be an important driver of the decline in CUE at higher latitudes. Our model suggests the quantitative contribution of ECM to soi! carbon to be less important but any possible implications through litter quality remain to be assessed. The approach provides a simple proxy of ECM processes for regional C budget models and estimates.

Carbon storage and cycling in boreal forests—the largest terrestrial carbon store—is moderated by complex interactions between trees and soil microorganisms. However, existing methods limit our ability to predict how changes in environmental conditions will alter these associations and the essential ecosystem services they provide. To address this, we developed a metatranscriptomic approach to analyze the impact of nutrient enrichment on Norway spruce fine roots and the community structure, function, and tree–microbe coordination of over 350 root-associated fungal species. In response to altered nutrient status, host trees redefined their relationship with the fungal community by reducing sugar efflux carriers and enhancing defense processes. This resulted in a profound restructuring of the fungal community and a collapse in functional coordination between the tree and the dominant Basidiomycete species, and an increase in functional coordination with versatile Ascomycete species. As such, there was a functional shift in community dominance from Basidiomycetes species, with important roles in enzymatically cycling recalcitrant carbon, to Ascomycete species that have melanized cell walls that are highly resistant to degradation. These changes were accompanied by prominent shifts in transcriptional coordination between over 60 predicted fungal effectors, with more than 5,000 Norway spruce transcripts, providing mechanistic insight into the complex molecular dialogue coordinating host trees and their fungal partners. The host–microbe dynamics captured by this study functionally inform how these complex and sensitive biological relationships may mediate the carbon storage potential of boreal soils under changing nutrient conditions.

Fertilization with nitrogen (N)-rich compounds leads to increased growth, but may compromise phenology and winter survival of trees in boreal regions. During autumn, N is remobilized from senescing leaves and stored in other parts of the tree to be used in the next growing season. However, the mechanism behind the N fertilization effect on winter survival is not well understood and it is unclear how N levels or forms modulate autumn senescence. We performed fertilization experiments and showed that treating Populus saplings with high or low levels of inorganic nitrogen resulted in a delay in senescence. In addition, by using precise delivery of solutes into the xylem stream of Populus trees in their natural environment, we found that delay of autumn senescence was dependent on the form of N administered: inorganic N (NO3−1) delayed senescence but amino acids (Arg, Glu, Gln, and Leu) did not. Metabolite profiling of leaves showed that the levels of tricarboxylic acids (TCA), arginine catabolites (ammonium, ornithine), glycine, glycine-serine ratio and overall carbon-to-nitrogen (C/N) ratio were affected differently by the way of applying NO3−1 and Arg treatments. In addition, the onset of senescence did not coincide with soluble sugar accumulation in any of the treatments. Taken together, metabolomic rearrangement under different N forms or experimental setups could modulate senescence process, but not initiation and progression in Populus. We propose that the different regulation of C and N status through direct molecular signaling of NO3−1 could account for the contrasting effects of NO3−1 and Arg on senescence.One sentence summaryNitrate, administered by precision fertilization through injection into the trunk, may delay autumn senescence and change metabolism in Populus leaves, while the same amount of amino acids does not have the same effect.

Soil extracellular enzyme activity (EEA) represents a critical bottleneck in the release of bioavailable nutrients from organic materials. However, quantifying spatial and temporal dynamics of EEA remains challenging. Techniques which measure the activity of, or directly sample free enzymes in situ may assist in understanding the short-term exoproteomic responses of microbes and roots to substrates, but few tools exist to explore EEA with minimal disturbance. We explore the potential of in situ microdialysis to directly sample soil enzymes, measuring their activity using a modified enzyme assay. We hypothesise that the technique's bias towards free solutes will also allow differentiation of free and stabilised enzyme pools. As little is known about the efficiency of microdialysis to sample enzymes from soil, recovery of a protease standard was quantified from solution and soil, finding that enzyme recovery is hindered at lower soil moisture contents. We further measured the response of native protease activity after the addition of soybean litter to clay and sandy soils, finding microdialysis observed greater EEA in litter-amended treatments than controls in both soil types. In comparison, EEA as measured by conventional extraction-incubation methods was only greater in amended clay soils. In a final experiment, hydrolytic enzyme activity of free and stabilised clay soil fractions were estimated using microdialysis. Free enzymes contributed 9% of total hydrolytic activity in soil without litter, increasing to 46% in litter-amended soil, suggesting fresh litter promoted a transient increase in the production of free exoenzymes by soil microbes. In contrast, the addition of litter had no significant effect on stabilised EEA. In spite of the obvious challenges involved in applying microdialysis as a method for soil protein sampling, this method offers new possibilities for investigating challenging spatial and temporal aspects of enzyme dynamics and protein availability in soils.

The boreal forest is an important global carbon (C) sink. Since low soil nitrogen (N) availability is commonly a key constraint on forest productivity, the prevalent view is that increased N input enhances its C sink-strength. This understanding however relies primarily on observations of increased aboveground tree biomass and soil C stock following N fertilization, whereas empirical data evaluating the effects on the whole ecosystem-scale C balance are lacking. Here we use a unique long-term experiment consisting of paired forest stands with eddy covariance measurements to explore the effect of ecosystem-scale N fertilization on the C balance of a managed boreal pine forest. We find that the annual C uptake (i.e. net ecosystem production, NEP) at the fertilized stand was 16 +/- 2% greater relative to the control stand by the end of the first decade of N addition. Subsequently, the ratio of NEP between the fertilized and control stand remained at a stable level during the following five years with an average NEP to N response of 7 & PLUSMN; 1 g C per g N. Our study reveals that this non-linear response of NEP to long-term N fertilization was the result of a cross-seasonal feedback between the N-induced increases in both growing-season C uptake and subsequent winter C emission. We further find that one decade of N addition altered the sensitivity of ecosystem C fluxes to key environmental drivers resulting in divergent responses to weather patterns. Thus, our study highlights the need to account for ecosystem-scale responses to perturbations to improve our understanding of nitrogen-carbon-climate feedbacks in boreal forests.

Boreal trees are capable of taking up organic nitrogen (N) as effectively as inorganic N. Depending on the abundance of soil N forms, plants may adjust physiological and morphological traits to optimize N uptake. However, the link between these traits and N uptake in response to soil N sources is poorly understood. We examined Pinus sylvestris L. seedlings’ biomass growth and allocation, transpiration and N uptake in response to additions of organic N (the amino acid arginine) or inorganic N (ammonium nitrate). We also monitored in situ soil N fluxes in the pots following an addition of N, using a microdialysis system. Supplying organic N resulted in a stable soil N flux, whereas the inorganic N resulted in a sharp increase of nitrate flux followed by a rapid decline, demonstrating a fluctuating N supply and a risk for loss of nitrate from the growth medium. Seedlings supplied with organic N achieved a greater biomass with a higher N content, thus reaching a higher N recovery compared with those supplied inorganic N. In spite of a higher N concentration in organic N seedlings, root-to-shoot ratio and transpiration per unit leaf area were similar to those of inorganic N seedlings. We conclude that enhanced seedlings’ nutrition and growth under the organic N source may be attributed to a stable supply of N, owing to a strong retention rate in the soil medium.

Understanding how plant water uptake interacts with acquisition of soil nitrogen (N) and other nutrients is fundamental for predicting plant responses to a changing environment, but it is an area where models disagree. We present a novel isotopic labelling approach which reveals spatial patterns of water and N uptake, and their interaction, by trees. The stable isotopes 15 N and 2 H were applied to a small area of the forest floor in stands with high and low soil N availability. Uptake by surrounding trees was measured. The sensitivity of N acquisition to water uptake was quantified by statistical modelling. Trees in the high-N stand acquired twice as much 15 N as in the low-N stand and around half of their N uptake was dependent on water uptake (2 H enrichment). In contrast, in the low-N stand there was no positive effect of water uptake on N uptake. We conclude that tree N acquisition was only marginally dependent on water flux toward the root surface under low N conditions but under high-N conditions, the water-associated N uptake was substantial. The results suggest a fundamental shift in N acquisition strategy under high-N conditions.

Scots pine seedlings were grown from sterilized seeds in oven sterilized sand. Half of the seedlings were inoculated with the mycorrhizal fungus Pisolithus arhizus (Pers.) Rausch (syn. Pisolithus tinctorius) while the rest were not. Seedlings were raised in a growth chamber for 61 d and supplied with a complete nutrient solution with NH

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There is a growing body of evidence that plants uptake a monumental amount of organic forms of nitrogen (N) like amino acids in addition to those in inorganic forms. An amino acid-based fertiliser has been shown to improve seedling development and commercialised. Boreal forests store a substantial amount of carbon (C) in the soil and this is widely known to be further enhanced by the addition of inorganic nitrogen fertiliser via hampered decomposition. However, very little is known about how amino acid-based fertiliser influences C/N cycling in the boreal soils. The organic forms of N supply not only nitrogen but also carbon. If the previously demonstrated suppression of SOM decomposition is owing to altered C:N ratios in substrates, the amino acid-based fertiliser may not have as pronounced effects on the soil as the inorganic fertiliser. We have examined the impacts of the organic fertiliser (100 kg N and 130 kg C ha-1 year-1)—arginine—on the chemical composition of soil organic matter in a boreal forest in comparison to non-fertilised, inorganic fertilised (ammonium-nitrate) and C-controlled inorganic fertilised (sucrose + ammonium-nitrate) conditions. The soil organic matter was characterised using two metrics: pyrolysis GC/MS and 13C solid-state nuclear magnetic resonance (NMR), combined with enzymological and metagenomic analysis.We will be presenting the results following 4-year of the fertiliser treatments. Preliminary results have shown that there is limited evidence that the fertiliser treatments alter soil C/N cycing in four years. Nevertheless, the chemical composition in SOM under the organic fertiliser condition was similar to that under C-controlled compared to inorganic fertiliser treatment.

Reliable manipulation of soil organic matter (SOM) – a necessity for optimal land management – is constrained by our limited mechanistic understanding of SOM formation. Here we add to existing frameworks a novel mechanistic element that may underpin SOM dynamics, based on evolutionary-ecological rather than chemical or physical limitations to decomposition. We argue that decomposition of some substrates may be ecologically constrained in mycelial fungi, evolutionarily constrained in co-operating bacteria, and geometrically constrained in unicellular microbes. We describe and test a mathematical model based on our framework, providing a proof-of-concept that substrate can plausibly be spared decomposition and accumulate even when it is physically and chemically accessible. Our framework can explain a variety of SOM dynamics, including priming and the suppression of decomposition by nitrogen addition, as well as the typical composition of SOM. An augmented mechanistic framework for understanding SOM dynamics can help guide targeted empirical study, which in turn can contribute to more optimised land management.

Ectomycorrhizal symbiosis is omnipresent in boreal forests, where it is assumed to benefit plant growth. However, experiments show inconsistent benefits for plants and volatility of individual partnerships, which calls for a re-evaluation of the presumed role of this symbiosis. We reconcile these inconsistencies by developing a model that demonstrates how mycorrhizal networking and market mechanisms shape the strategies of individual plants and fungi to promote symbiotic stability at the ecosystem level. The model predicts that plants switch abruptly from a mixed strategy with both mycorrhizal and nonmycorrhizal roots to a purely mycorrhizal strategy as soil nitrogen availability declines, in agreement with the frequency distribution of ectomycorrhizal colonization intensity across a wide-ranging data set. In line with observations in field-scale isotope labeling experiments, the model explains why ectomycorrhizal symbiosis does not alleviate plant nitrogen limitation. Instead, market mechanisms may generate self-stabilization of the mycorrhizal strategy via nitrogen depletion feedback, even if plant growth is ultimately reduced. We suggest that this feedback mechanism maintains the strong nitrogen limitation ubiquitous in boreal forests. The mechanism may also have the capacity to eliminate or even reverse the expected positive effect of rising CO2 on tree growth in strongly nitrogen-limited boreal forests.

Plant processes, ranging from photosynthesis through production of biomaterials to environmental sensing and adaptation, can be used in technology via integration of functional materials and devices. Previously, plants with integrated organic electronic devices and circuits distributed in their vascular tissue and organs have been demonstrated. To circumvent biological barriers, and thereby access the internal tissue, plant cuttings were used, which resulted in biohybrids with limited lifetime and use. Here, we report intact plants with electronic functionality that continue to grow and develop enabling plant-biohybrid systems that fully maintain their biological processes. The biocatalytic machinery of the plant cell wall was leveraged to seamlessly integrate conductors with mixed ionic-electronic conductivity along the root system of the plants. Cell wall peroxidases catalyzed ETE-S polymerization while the plant tissue served as the template, organizing the polymer in a favorable manner. The conductivity of the resulting p(ETE-S) roots reached the order of 10 S cm(-1) and remained stable over the course of 4 weeks while the roots continued to grow. The p(ETE-S) roots were used to build supercapacitors that outperform previous plant-biohybrid charge storage demonstrations. Plants were not affected by the electronic functionalization but adapted to this new hybrid state by developing a more complex root system. Biohybrid plants with electronic roots pave the way for autonomous systems with potential applications in energy, sensing and robotics. Funding Agencies|European UnionEuropean Commission [800926, 838171]; Swedish Research CouncilSwedish Research CouncilEuropean Commission [VR-2017-04910]; Knut and Alice Wallenberg FoundationKnut & Alice Wallenberg Foundation; Wallenberg Wood Science Center [KAW 2018.0452]; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University [2009-00971]; European Research Council (ERC)European Research Council (ERC)European Commission [834677]

Boreal forests store approximately 470 Pg of carbon (C) in the soil, and rates of soil C accumulation are significantly enhanced by long-term nitrogen (N) enrichment. Dissecting the compositional profile of soils could help better understand the potential mechanisms driving changes in C cycling under enriched N conditions. We examined the impacts of long-term N addition on the chemical composition of soil organic matter (SOM) in a mature boreal forest. Two large experimental plots (15 ha each) were established: a control and a fertilised plot. The latter received NH4NO3 fertilizer at an average rate of 75 kg N ha−1 year−1 for 12 years. While the centre of this plot received the prescribed amounts of fertilizer, the year-to-year variation in distribution of fertilizer around the designated edges of the plot created a gradient in N-loading. Along this gradient, a compositional shift in SOM in the organic horizon was assessed using two methods: pyrolysis-gas chromatography/mass spectrometry (GC/MS) and solid-state 13C nuclear magnetic resonance spectroscopy (13C NMR). Both of these methods revealed that the chemical composition of SOM changed with increasing N loading, with an increased fraction of lignin derivatives (i.e., aromatic, methoxy/N-alkyl C) relative to that of carbohydrate (i.e., O-alkyl C), accompanied by increased soil C mass (kg m−2) at the fertilised plot. Also, the relative abundance of N compounds in the pyrolysis products increased with the N loading, mainly due to increased methyl N-acetyl-α-D-glucosaminide in the F/H horizon, plausibly of microbial origin. Microbial N processing likely contributed to soil accumulation of fertilizer-derived N. Our results corroborate the hypothesis that addition of inorganic N suppresses enzymatic white-rot decomposition relative to non-enzymatic brown-rot oxidation. Taken together, our study suggests that N enrichment leads to a selective accumulation of lignin-derived compounds and points to a key role of such compounds for N-induced SOM accumulation.

Forests pass water and carbon through while converting portions to streamflow, soil organic matter, wood production, and other ecosystem services. The efficiencies of these transfers are but poorly quantified. New theory and new instruments have made it possible to use stable isotope composition to provide this quantification of efficiencies wherever there is a measurable difference between the branches of a branchpoint. We present a linked conceptual model that relies on isotopes of hydrogen, carbon, and oxygen to describe these branchpoints along the pathway from precipitation to soil and biomass carbon sequestration and illustrate how it can be tested and generalized.Plain Language Summary The way a forest works can be described in terms of carbon and water budgets, which describe the ways that carbon and water flow through the forest. The paths of such flows are frequently branched and the branches are often different in their stable isotope composition. This means that stable isotopes can be used to describe the branching events. We present isotopic methods of quantifying several such events, then link them in a chain that begins with the evaporation of water and ends with biomass production.

Important structures and functions within living organisms rely on naturally fluorescent polymeric molecules such as collagen, keratin, elastin, resilin, or lignin. Theoretical physics predict that fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls and one of the most abundant components of wood. Lignin is composed of different types of phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured wood cell wall composition by classical analytical methods in the wood cell walls of these trees. Using statistical modelling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm plants. Finally, we show how quantitative measurements of lignin chemical composition by FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

Nitrogen (N) added through atmospheric deposition or as fertilizer in boreal forests may alter their carbon (C) sequestration potential and sensitivity to climatic changes. While previous studies have primarily explored the responses of individual ecosystem components such as stem biomass production and soil carbon changes following N addition, the long-term impacts of N addition on the ecosystem-scale C balance of boreal forests still remain unclear. Here, we use data from eddy-covariance measurements in a fertilized Scots pine (Pinus sylvestris L.) forest (i.e. 16 ha receiving 100 and 50 kg N ha-1 yr-1 since 2006 and 2012, respectively) and an adjacent unfertilized control stand in boreal Sweden to investigate how one decade of N addition affected the net ecosystem productivity (NEP), gross primary production (GPP) and ecosystem respiration (ER) over five fertilization years (2015-2019). Results showed that N fertilization increased GPP in all five years with by 18% at average to 1183±41 g C m-2 yr-1 in the N-fertilized stand compared to 1003±56 g C m-2 yr-1 in the control stand. ER was also increased from 744±29 g C m-2 yr-1 in the control stand to 875±37 g C m-2 yr-1 in the fertilized stand. As a result, fertilization increased NEP from 259±28 g C m-2 yr-1 in the control stand to 308±20 g C m-2 yr-1 in the N-fertilized stand. Our results further suggested that the annual NEP was similar between stands during years with normal weather conditions (2015-2016) while NEP diverged due to a larger reduction in the control stand in years with environmental constraints (i.e. a cool summer in 2017 and droughts in 2018 and 2019). These findings indicate that enhanced N input to boreal forests increases and stabilizes their C sequestration potential under future climate conditions.

Plant root exudates are believed to increase root capture of nutrients (including nitrogen) by encouraging development of a rhizosphere root community, and providing them with an energy source to facilitate degradation of litter and soil organic matter. However, observing the consequences of root exudation on nutrient cycling and microbial activity is challenging with current methods, given the small scales involved. We investigated the effect of root exudation on nitrogen (N) availability by simulating root exudation with microdialysis. This novel technique enables continuous release of synthetic solutions of root exudates via diffusion in situ in soil by using a root-sized permeable membrane. Importantly, it also allows for simultaneously monitoring the effects on inorganic N fluxes. To emulate growth of a root tip through a specific soil region, sucrose was released for seven days before substituting sucrose with water for a further 7 days. We investigated boreal forest soils with and without litter amendments (ground pea shoots) to attain different C/N ratios and we used two rates of exudation by retrodialysing with either 0.5 or 5 mM sucrose solution. We observed that pea litter promoted significant N immobilisation, along with greater rates of sucrose release from microdialysis probes - peaking at 90.7 ± 8 µg sucrose m-2 s-1 using the 5 mM sucrose solution after three days. This suggests that greater root exudation may be driven by microbial demand for both C and N, with no short-term nutritional benefit for plant roots, even after exudation has ceased within a specific soil region. Glucose and fructose fluxes (breakdown products of sucrose) were also greatest in the litter treatment, indicating enzyme activity was promoted by the availability of both sucrose and litter. CO2 respiration measurements indicated significant differences between litter and control soils, but there was no detectable effect of sucrose exudation, suggesting that the small amounts of C supplied and the limited area influenced by the diffusion of sucrose had little impact on overall microcosm respiration. We conclude that short-term C exudation presented no immediate benefit for plant nutrition in our experiment. Future studies can benefit from using microdialysis to investigate the influence of more complex root exudate solutions, as well as the mechanistic roles of transpiration-induced mass flow on plant N availability in the rhizosphere.

Root exudates are thought to promote nitrogen (N) availability via rhizosphere interactions, but empirical evidence is difficult to obtain given the scale and temporary nature of these processes. Microdialysis has potential to simulate root exudation patterns and quantify the effects on N availability simultaneously, but this has so far not been attempted. In a conceptual root exudation study, we used sucrose as a simple C source to investigate if microdialysis could detect the effects of continuous localised C supply on soil inorganic N fluxes. Through retrodialysis we released sucrose and simultaneously monitored diffusive soil N fluxes over one week, followed by a further seven days without sucrose. Based on current understanding of rhizosphere N dynamics, we hypothesised that N fluxes are inversely related to sucrose release, and upon ceasing release, N fluxes would increase. Using a 5 mM sucrose perfusate, C releases resulted in decreased N fluxes, but contrary to our hypothesis, N fluxes did not increase after ceasing sucrose release (c.f. control soil). Diffusive sucrose efflux from microdialysis probes increased in soils amended with N-rich litter suggesting that microbial activity and associated sucrose consumption altered sucrose concentration gradients. The fluxes of sucrose breakdown products glucose and fructose were greatest in litter treatments receiving sucrose, indicative of increased invertase activity in the presence of fresh organic matter. In the short term (days), sucrose release did not prompt an increase in inorganic N availability, possibly because of stimulated microbial growth and increased N demand under C-rich conditions. Our study confirms that microdialysis allows time-sensitive insight into the dynamic interactions of carbon and N in the rhizosphere.

Increased soil organic matter decomposition with increasing temperature has been hypothesized to enhance soil nitrogen availability, consequently stimulating forest biomass production and offsetting decomposition-induced soil carbon losses1–5. This projection, however, is based on evidence gathered from short-term studies (≤10 years)2,3,5. The key question for carbon sequestration is whether such responses are transient or persist over long forest rotation periods. Here we report on biomass production in a typical nitrogen-limited boreal Picea abies forest, exposed to 18 years of soil warming manipulation (+5 °C) at a plot scale (100 m2). We show that two decades of soil warming elicited only short-duration growth responses, thus not significantly increasing aboveground biomass accumulation. Furthermore, in combination with published work from this forest, our results suggest that increased decomposition is slight and ephemeral, and increased fine root production and turnover in deeper soil may be greater than increased decomposition, netting slightly more biomass, perhaps conserving the soil carbon stock. Thus, this long-term study does not support the notion that the projected increase in soil temperatures will cause either an increased carbon loss with decomposition or a compensatory growth increase from nitrogen mineralization. Nearly two decades of data from a boreal forest soil warming experiment (+5 °C) show no significant increase in aboveground biomass accumulation beyond an initial transitory response.

Interactions between Norway spruce trees and bacteria and fungi in nutrient limited boreal forests can be beneficial for tree growth and fitness. Tree-level effects of anthropogenic nutrient addition have been well studied, however understanding of the long-term effects on the associated microbiota is limited. Here, we report on the sensitivity of microbial community composition to the growing season and nutrient additions. Highthroughput sequencing of the bacterial 16S rRNA gene and fungal ITS1 region was used to characterise changes in the microbial community after application of a complete mineral nutrient mixture for five and 25 years. The experiment was conducted using the Flakaliden forest research site in northern boreal Sweden and included naturally low nutrient control plots. Needle and fine root samples of Norway spruce were sampled in addition to bulk soil during one growing season to provide comprehensive insight into phyllosphere and belowground microbiota community changes. The phyllosphere microbiota was compositionally distinct from the belowground communities and phyllosphere diversity increased significantly over the growing season but was not influenced by the improved nutrient status of the trees. In both root and soil samples, alpha diversity of fungal, in particular ectomycorrhizal fungi (EMF), and bacterial communities increased after long-term nutrient optimisation, and with increasing years of treatment the composition of the fungal and bacterial communities changed toward a community with a higher relative abundance of nitrophilic EMF and bacterial species but did not cause complete loss of nitrophobic species from the ecosystem. From this, we conclude that 25 years of continuous nutrient addition to a boreal spruce stand increased phylotype richness and diversity of the microbiota in the soil, and at the root-soil interface, suggesting that long-term anthropogenic nutrient inputs can have positive effects on belowground biodiversity that may enhance ecosystem robustness. Future studies are needed to assess the impact of these changes to the microbiota on ecosystem carbon storage and nitrogen cycling in boreal forests. Bio4Energy

The forest hydrologic cycle is expected to have important feedback responses to climate change, impacting processes ranging from local water supply and primary productivity to global water and energy cycles. Here, we analyzed water budgets of pine forests worldwide. We first estimated local water balance of forests dominated by two wide-ranging species: Pinus taeda (36 °N) and Pinus sylvestris (64 °N). In these stands, growing season evapotranspiration (ET) was largely insensitive to inter-annual variation of precipitation (P), consistent with the insensitivity of canopy transpiration to P. Extending the analyses to include published data from 117 studies on 27 pine species, we found that pine forests annually use ∼66% ± 17% (SD) of P as ET, regardless of climatic regime, leaving a third of P as runoff to downstream aquatic ecosystems and users. However, during the growing season, pine forests used more water as ET than P in regions where P ≤ 326 ± 39 (SE) mm. Forests in regions of low growing season P exist in their current state only where the rooting depth is sufficient to supply trees with water from soil storage in addition to P, and these forests are likely to support only ephemeral streams that dry down during the growing season. Thus, globally, water use by pine forests is adapted to mean annual P, but shows a limited capacity to respond to inter-annual variability in P. Forests with a small buffer of growing season water availability (P + soil water storage - ET), are likely to be most sensitive to variation in P regimes, changing canopy leaf area, tree density, and species composition depending on the degree, direction and persistence of the change in P.

Nitrogen (N) addition causes rapid accumulation of carbon (C) in the soils of boreal forests. The C accumulation has been attributed to an increase in C supply to the soil, to a decrease in mineralization of organic C to CO2, or some combination of the two. We sought to quantify the proportional causes in a case study in a boreal Scots pine forest with or without annual N addition (at 50–100 kg N ha−1 yr−1). We continuously measured soil-surface CO2 exchange with large chambers (20-m2 surface area) over three growing seasons and derived flux rates from snowpack CO2 profiles during the winter. Models were used to disaggregate the CO2-exchange data into autotrophic and heterotrophic components. We also measured litterfall and inferred total belowground carbon flux (TBCF). We observed annual soil C accumulation to be higher by 104 g C m−2 yr−1 at the fertilized (F) plot compared to the unfertilized reference (R) stand. Total annual C supply to soil (Aboveground litterfall + TBCF) was not increased by the N addition despite a substantial increase in litterfall (+64 g C m−2 yr−1). Instead the sum decreased slightly, by 52 g C m−2 yr−1, because of a larger reduction in TBCF. This reduction in soil C supply led us to conclude that the soil C accumulation was entirely due to inhibited substrate mineralization. We speculate that the nitrogen increased soil C by inhibiting heterotrophy, specifically that portion of heterotrophy that is primed by autotrophic carbon.

Mixed forests have been recommended to replace monocultures, often being more productive and resilient ecosystems. Those benefits of species mixing have been suggested to be attributed to lower competition (above- and belowground) due to potential separation of resource acquisition strategies, yet a mechanistic understanding of belowground processes has largely been missing. We applied an isotopic-labelling technique using 15N and 2H at ~5 cm soil depth to study acquisition of two important resources, nitrogen (N) and water, in a replicated field experiment including mature Scots pine and Norway spruce monocultures and their mixture in boreal Sweden. Based on the isotopic data, we modelled distance-dependent N and water uptake and described the horizontal reach of active rooting areas around trees. The active root area for both N and water uptake in monocultures of both species was approximately 6–10 m2. Substantially wider areas were observed inside the mixture for N acquisition (27 m2 for pine and 21 m2 for spruce). Water was mainly acquired from within a 12–15 m2 area in mixture. The mixture and the pine monocultures exhibited similar recovery of the added 15N (3.8–4.5%) and its uptake per unit of foliage mass. The recovery of 15N for the spruce monocultures was generally lower (2.1%) but no difference was noted in the uptake per unit of foliage mass between spruce monoculture and mixture. No differences were found for recovery of 2H between the stand types. Mixing pine and spruce did not improve the resource uptake in comparison with the estimated theoretical mixture (according to the normalized basal area in monocultures). However, the mixing revealed significant differences between tree species whereas pines inside the mixture recovered 11.3-fold more 15N than accompanying spruce. Root overlap of 3–11 trees on 1 m2 was observed in all stand types, far exceeding the degree of canopy overlap. We discuss this overlap in terms of competition between individual trees. Spruces have proportionally larger horizontal rooting area relative to their aboveground size than pines. Scots pine and Norway spruce mixing in this type of boreal forest does not significantly enhance resource acquisition but leads to compensatory partitioning of growth, suggesting fierce interspecific competition, rather than niche separation.

Nitrogen (N) addition effects on boreal forest ecosystem are influenced by an interplay between N-form and N-dose. We hypothesize that trees take up organic N more efficiently than inorganic N and that unwanted side-effects of organic N are smaller. We predicted that 1) the tree growth response to arginine (ARG) addition is larger than to ammonium-nitrate (AN) and, 2) understory vegetation and ectomycorrhizal (EcM) changes following ARG addition are smaller than following AN addition. We investigated the effects of AN and ARG addition (50 and 150 kg N ha−1) during five years on tree growth, understory vegetation and EcM fungi in a Pinus sylvestris L. forest (c 50 years old) in northern Sweden. N addition increased tree growth and changed understory vegetation composition with few significant differences between AN and ARG. Differences in responses mainly occurred for the bryophyte Pleurozium schreberi which decreased more from ARG, and for EcM sporocarps, which sharply declined from AN, but not from ARG. We found very few differences in responses between AN and ARG addition with the exception of EcM and bryophytes. These species groups have several key functions in boreal forests and the differences in responses merits further investigations.

Microdialysis is a technique that can be used to sample fluxes of nitrogen (N) in soils with minimal disturbance. To advance our understanding of the technique and improve N recovery, we compared a common membrane type (10 x 0:5 mm probe length and width, 20 kDa molecular weight cut-off; MWCO) with alternative length and MWCO configurations (30 mm; and 100 kDa MWCO). We hypothesised that the alternative membranes would improve recovery of low molecular weight N via increased surface area and membrane pore size. The test environments, sampled at fixed pump flow rates, were: (i) stirred 100 mu M N standard solution containing organic (amino acid) and inorganic (ammonium, nitrate) N; (ii) soil spiked with 100 mu M standard N solution; and (iii) in situ boreal forest soil. In general, long membranes recovered more N, but the magnitude of improved recovery varied with test environment. Long membranes recovered more inorganic N regardless of flow rate, except ammonium in stirred solution, where length had no effect at slow flow rates. Long membranes also recovered more organic N from stirred solution regardless of flow rate, and recovered most N at slow flow rates in spiked soil. Longer membranes recovered more amino acids in situ in forest soil, with improved resolution of individual amino acids, but were biased towards soluble, mobile forms. MWCO did not affect N recoveries, indicating that in the test conditions, membrane length had greater control than pore size. We discuss the bottlenecks of microdialysis application in soil research and conclude that optimised membrane configurations will advance its use as a tool for quantifying nutrient fluxes in soils. (C) 2017 Elsevier Ltd. All rights reserved.

We report results from long-term simulated external nitrogen (N) input experiments in three northern Pinus sylvestris forests, two of moderately high and one of moderately low productivity, assessing effects on annual net primary production (NPP) of woody mass and its interannual variation in response to variability in weather conditions. A sigmoidal response of wood NPP to external N inputs was observed in the both higher and lower productivity stands, reaching a maximum of ~65% enhancement regardless of the native site productivity, saturating at an external N input of 4-5 g N·m-2 ·yr-1 . The rate of increase in wood NPP and the N response efficiency (REN , increase in wood NPP per external N input) were maximized at an external N input of ~3 g N·m-2 ·yr-1 , regardless of site productivity. The maximum REN was greater in the higher productivity than the lower productivity stand (~20 vs. ~14 g C/g N). The N-induced enhancement of wood NPP and its REN were, however, markedly contingent on climatic variables. In both of the higher and lower productivity stands, wood NPP increased with growing season precipitation (P), but only up to ~400 mm. The sensitivity of the response to P increased with increasing external N inputs. Increasing growing season temperature (T) somewhat increased the N-induced drought effect, whereas decreasing T reduced the drought effect. These responses of wood NPP infused a large temporal variation to REN , making the use of a fixed value unadvisable. Based on these results, we suggest that regional climate conditions and future climate scenarios should be considered when modeling carbon sequestration in response to N deposition in boreal P. sylvestris, and possibly other forests.

Several studies have suggested that CO2 transport in the transpiration stream can considerably bias estimates of root and stem respiration in ring-porous and diffuse-porous tree species. Whether this also happens in species with tracheid xylem anatomy and lower sap flow rates, such as conifers, is currently unclear. We infused 13C-labelled solution into the xylem near the base of two 90-year-old Pinus sylvestris L. trees. A custom-built gas exchange system and an online isotopic analyser were used to sample the CO2 efflux and its isotopic composition continuously from four positions along the bole and one upper canopy shoot in each tree. Phloem and needle tissue 13C enrichment was also evaluated at these positions. Most of the 13C label was lost by diffusion within a few metres of the infusion point indicating rapid CO2 loss during vertical xylem transport. No 13C enrichment was detected in the upper bole needle tissues. Furthermore, mass balance calculations showed that c. 97% of the locally respired CO2 diffused radially to the atmosphere. Our results support the notion that xylem CO2 transport is of limited magnitude in conifers. This implies that the concerns that stem transport of CO2 derived from root respiration biases chamber-based estimates of forest carbon cycling may be unwarranted for mature conifer stands.

Adding nitrogen to boreal forest ecosystems commonly increases gross primary production (GPP). The effect of nitrogen addition on ecosystem GPP is convoluted due to the impacts of and interactions among leaf scale photosynthetic productivity, canopy structure, site fertility, and environmental constraints. We used a unique controlled nitrogen fertilisation experiment combined with eddy covariance measurements and the calibration of a LUE-based (light use efficiency) photosynthetic production model in order to reveal differences in photosynthetic capacity due to nitrogen addition. A systematically designed soil moisture survey was conducted to characterise the within-site spatial heterogeneity and validate the difference of water stress between fertilised and control sites. The canopy photosynthetic light responses and environmental constraints were evaluated using an inverse modelling approach. We found that nitrogen fertilisation elevated ecosystem GPP by 24% according to model simulations. This was caused by increases in ecosystem light interception (through an increase in leaf area index (LAI)) and LUE by 7% and 17%, respectively. Nitrogen addition increased canopy potential LUE for both low and high photosynthetic photon flux density (PPFD) conditions. The calculations of leaf area and light interception indicated that the understorey vegetation contributed 9% of ecosystem GPP in the fertilised site and 7% in the control site when assuming a same LUE for trees and shrubs. The constraint arising from atmospheric water demand, rather than soil water stress, was the dominating control of the intra- and inter-annual GPP variations. The uncertainty propagated from soil moisture data is negligible for GPP predictions, but influential in the inference on the severity of the drought. This study demonstrates the combination of the controlled field experiment with the inverse modelling approach provides a powerful tool to quantitatively describe and disaggregate N addition effects on forest ecosystem GPP.

Although organic nitrogen (N) compounds are ubiquitous in soil solutions, their potential role in plant N nutrition has been questioned. We performed a range of experiments on Arabidopsis thaliana genetically modified to enhance or reduce root uptake of amino acids. Plants lacking expression of the Lysine Histidine Transporter 1 (LHT1) displayed significantly lower contents of 13 C and 15 N label and of U-13 C5 ,15 N2 L-glutamine, as determined by liquid chromatography-mass spectrometry when growing in pots and supplied with dually labelled L-glutamine compared to wild type plants and LHT1-overexpressing plants. Slopes of regressions between accumulation of 13 C-labelled carbon and 15 N-labelled N were higher for LHT1-overexpressing plants than wild type plants, while plants lacking expression of LHT1 did not display a significant regression between the two isotopes. Uptake of labelled organic N from soil tallied with that of labelled ammonium for wild type plants and LHT1-overexpressing plants but was significantly lower for plants lacking expression of LHT1. When grown on agricultural soil plants lacking expression of LHT1 had the lowest, and plants overexpressing LHT1 the highest C/N ratios and natural δ15 N abundance suggesting their dependence on different N pools. Our data show that LHT1 expression is crucial for plant uptake of organic N from soil.

Net uptake of carbon dioxide (CO2) was observed during the winter when using the eddy covariance (EC) technique above a ∼90-year-old Scots pine (Pinus sylvestris L.) stand in northern Sweden. This uptake occurred despite photosynthetic dormancy. This discrepancy led us to investigate the potential impact of decoupling of below- and above-canopy air mass flow and accompanying below-canopy horizontal advection on these measurements. We used the correlation of above- and below-canopy standard deviation of vertical wind speed (σw), derived from EC measurements above and below the canopy, as the main mixing criterion. We identified 0.33 m s−1 and 0.06 m s−1 as site-specific σw thresholds for above and below canopy, respectively, to reach the fully coupled state. Decoupling was observed in 45% of all cases during the measurement period (5.11.2014–25.2.2015). After filtering out decoupled periods the above-canopy mean winter NEE shifted from −0.52 μmol m−2 s−1 to a more reasonable positive value of 0.31 μmol m−2 s−1. None of the above-canopy data filtering criteria we tested (i.e., friction velocity threshold; horizontal wind speed threshold; single-level σw threshold) ensured sufficient mixing. All missed critical periods that were detected only by the two-level filtering approach. Tower-surrounding topography induced a predominant below-canopy wind direction and consequent wind shear between above- and below-canopy air masses. These processes may foster decoupling and below-canopy removal of CO2 rich air. To determine how broadly such a topographical influence might apply, we compared the topography surrounding our tower to that surrounding other forest flux sites worldwide. Medians of maximum elevation differences within 300 m and 1000 m around 110 FLUXNET forest EC towers were 24 m and 66 m, respectively, compared to 24 m and 114 m, respectively, at our site. Consequently, below-canopy flow may influence above-canopy NEE detections at many forested EC sites. Based on our findings we suggest below-canopy measurements as standard procedure at sites evaluating forest CO2 budgets.

Models predicting ecosystem carbon dioxide (CO2 ) exchange under future climate change rely on relatively few real-world tests of their assumptions and outputs. Here, we demonstrate a rapid and cost-effective method to estimate CO2 exchange from intact vegetation patches under varying atmospheric CO2 concentrations. We find that net ecosystem CO2 uptake (NEE) in a boreal forest rose linearly by 4.7 ± 0.2% of the current ambient rate for every 10 ppm CO2 increase, with no detectable influence of foliar biomass, season, or nitrogen (N) fertilization. The lack of any clear short-term NEE response to fertilization in such an N-limited system is inconsistent with the instantaneous downregulation of photosynthesis formalized in many global models. Incorporating an alternative mechanism with considerable empirical support - diversion of excess carbon to storage compounds - into an existing earth system model brings the model output into closer agreement with our field measurements. A global simulation incorporating this modified model reduces a long-standing mismatch between the modeled and observed seasonal amplitude of atmospheric CO2 . Wider application of this chamber approach would provide critical data needed to further improve modeled projections of biosphere-atmosphere CO2 exchange in a changing climate.