The effect of nitrogen source and levels on hybrid aspen tree physiology and wood formation.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density. An optimal level of nitrogen availability was identified where increases in biomass accumulation outweighed decreases in wood density. None of these responses depended on the source of nitrogen except for shoot biomass accumulation, which was stimulated more by treatments complemented with nitrate than by ammonium alone or the organic source arginine. The most striking difference between the nitrogen sources was the effect on lignin composition, whereby the abundance of H-type lignin increased only in the presence of nitrate. The differential effect of nitrate is possibly related to the well-known role of nitrate as a signaling compound. RNA-sequencing revealed that while the lignin-biosynthetic genes did not significantly (FDR <0.01) respond to added NO3 - , the expression of several laccases, catalysing lignin polymerization, was dependent on N-availability. These results reveal a unique role of nitrate in wood formation and contribute to the knowledge basis for decision-making in utilizing hybrid aspen as a bioresource.

Forest inventory tree core archive reveals changes in boreal wood traits over seven decades.

Boreal forests play an important role in the global carbon (C) cycle, and there is great interest in understanding how they respond to environmental change, including nitrogen (N) and water limitation, which could impact future forest growth and C storage. Utilizing tree cores archived by the Swedish National Forest Inventory, we measured stemwood traits, including stable N and C isotope composition which provides valuable information related to N availability and water stress, respectively, as well as N and C content, and C/N ratio over 1950-2017 in two central Swedish counties covering an area of ca. 55,000 sq. km (n = 1038). We tested the hypothesis that wood traits are changing over time, and that temporal patterns would differ depending on alternative dendrochronological reconstruction methods, i.e. the commonly applied "single tree method" (STM) or a conceptually stronger "multiple tree method" (MTM). Averaged across all MTMs, our data showed that all five wood traits for Picea abies and Pinus sylvestris changed over time. Wood δ15N strongly declined, indicating progressive nitrogen limitation. The decline in δ13C tracked the known atmospheric δ13CO2 signal, suggesting no change in water stress occurred. Additionally, wood N significantly increased, while C and C/N ratios declined over time. Furthermore, wood trait patterns sometimes differed between dendrochronological methods. The most notable difference was for δ15N, where the slope was much shallower for the STM compared to MTMs for both species, indicating that mobility of contemporary N is problematic when using the STM, resulting in substantially less sensitivity to detect historical signals. Our study indicates strong temporal changes in boreal wood traits and also indicates that the field of dendroecology should adopt new methods and archiving practices for studying highly mobile element cycles, such as nitrogen, which are critical for understanding environmental change in high latitude ecosystems.

Timing is everything - obtaining accurate measures of plant uptake of amino acids.

Plants are known to have the capacity to take up and utilise amino acids for growth. The significance of this uptake, however, remains elusive, partly due to methodological challenges and biological implications associated with acquiring and interpreting data. This study compared bulk stable isotope analysis and compound-specific liquid chromatography-mass spectrometry, two established methods for determining amino acid uptake. Root amino acid uptake was assayed using U-13 C5 -15 N2 -l-glutamine and axenically grown Arabidopsis thaliana. After 15-120 min of exposure, the content of intact glutamine measured in the roots was constant, whilst the 15 N and 13 C content increased over time, resulting in very different estimated uptake rates. The 13 C : 15 N ratio in roots declined with time, suggesting a loss of glutamine carbon of up to 15% within 120 min. The results presented indicate that, regardless of method used, time is a crucial factor when determining plant amino acid uptake. Due to post-uptake metabolism, compound-specific methods should primarily be used in experiments with a time frame of minutes rather than hours or days. Post-uptake metabolism in plants may account for significant loss of carbon, suggesting that it is not just pre-uptake metabolism by microbes that accounts for the 15 N-13 C mismatch reported in ecological studies, but also post-uptake metabolism in the plant.

Organic nitrogen enhances nitrogen nutrition and early growth of Pinus sylvestris seedlings.

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.

Novel Microdialysis Technique Reveals a Dramatic Shift in Metabolite Secretion during the Early Stages of the Interaction between the Ectomycorrhizal Fungus Pisolithus microcarpus and Its Host Eucalyptus grandis.

The colonisation of tree roots by ectomycorrhizal (ECM) fungi is the result of numerous signalling exchanges between organisms, many of which occur before physical contact. However, information is lacking about these exchanges and the compounds that are secreted by each organism before contact. This is in part due to a lack of low disturbance sampling methods with sufficient temporal and spatial resolution to capture these exchanges. Using a novel in situ microdialysis approach, we sampled metabolites released from Eucalyptus grandis and Pisolithus microcarpus independently and during indirect contact over a 48-h time-course using UPLC-MS. A total of 560 and 1530 molecular features (MFs; ESI- and ESI+ respectively) were identified with significant differential abundance from control treatments. We observed that indirect contact between organisms altered the secretion of MFs to produce a distinct metabolomic profile compared to either organism independently. Many of these MFs were produced within the first hour of contact and included several phenylpropanoids, fatty acids and organic acids. These findings show that the secreted metabolome, particularly of the ECM fungus, can rapidly shift during the early stages of pre-symbiotic contact and highlight the importance of observing these early interactions in greater detail. We present microdialysis as a useful tool for examining plant-fungal signalling with high temporal resolution and with minimal experimental disturbance.

Organic nitrogen nutrition: LHT1.2 protein from hybrid aspen (Populus tremula L. x tremuloides Michx) is a functional amino acid transporter and a homolog of Arabidopsis LHT1.

The contribution of amino acids (AAs) to soil nitrogen (N) fluxes is higher than previously thought. The fact that AA uptake is pivotal for N nutrition in boreal ecosystems highlights plant AA transporters as key components of the N cycle. At the same time, very little is known about AA transport and respective transporters in trees. Tree genomes may contain 13 or more genes encoding the lysine histidine transporter (LHT) family proteins, and this complicates the study of their significance for tree N-use efficiency. With the strategy of obtaining a tool to study N-use efficiency, our aim was to identify and characterize a relevant AA transporter in hybrid aspen (Populus tremula L. x tremuloides Michx.). We identified PtrLHT1.2, the closest homolog of Arabidopsis thaliana (L.) Heynh AtLHT1, which is expressed in leaves, stems and roots. Complementation of a yeast AA uptake mutant verified the function of PtrLHT1.2 as an AA transporter. Furthermore, PtrLHT1.2 was able to fully complement the phenotypes of the Arabidopsis AA uptake mutant lht1 aap5, including early leaf senescence-like phenotype, reduced growth, decreased plant N levels and reduced root AA uptake. Amino acid uptake studies finally showed that PtrLHT1.2 is a high affinity transporter for neutral and acidic AAs. Thus, we identified a functional AtLHT1 homolog in hybrid aspen, which harbors the potential to enhance overall plant N levels and hence increase biomass production. This finding provides a valuable tool for N nutrition studies in trees and opens new avenues to optimizing tree N-use efficiency.

Organic Nitrogen Uptake and Assimilation in Cucumis sativus Using Position-Specific Labeling and Compound-Specific Isotope Analysis.

Organic nitrogen is now considered a significant source of N for plants. Although organic management practices increase soil organic C and N content, the importance of organic N as a source of crop N under organic farming management systems is still poorly understood. While dual-labeled (13C and 15N) molecule methods have been developed to study amino acid uptake by plants, multiple biases may arise from pre-uptake mineralization by microorganisms or post-uptake metabolism by the plant. We propose the combination of different isotopic analysis methods with molecule isotopologues as a novel approach to improve the accuracy of measured amino acid uptake rates in the total N budget of cucumber seedlings and provide a better characterization of post-uptake metabolism. Cucumber seedlings were exposed to solutions containing L-Ala-1-13C,15N or U-L-Ala-13C3,15N, in combination with ammonium nitrate, at total N concentrations ranging from 0 to 15 mM N and at inorganic/organic N ratios from 10:1 to 500:1. Roots and shoots were then subjected to bulk stable isotope analysis (BSIA) by Isotope Ratio Mass Spectrometry (IRMS), and to compound-specific stable isotope analysis (CSIA) of the free amino acids by Gas Chromatography - Combustion - Isotope Ratio Mass Spectrometry (GC-C-IRMS). Plants exposed to a lower inorganic:organic N ratio acquired up to 6.84% of their N from alanine, compared with 0.94% at higher ratio. No 13C from L-Ala-1-13C,15N was found in shoot tissues suggesting that post-uptake metabolism of Ala leads to the loss of the carboxyl-C as CO2. CSIA of the free amino acids in roots confirmed that intact Ala is indeed taken up by the roots, but that it is rapidly metabolized. C atoms other than from the carboxyl group and amino-N from Ala are assimilated in other amino acids, predominantly Glu, Gln, Asp, and Asn. Uptake rates reported by CSIA of the free amino acids are nevertheless much lower (16-64 times) than those reported by BSIA. Combining the use of isotopologues of amino acids with compound-specific isotope analysis helps reduce the bias in the assessment of organic N uptake and improves the understanding of organic N assimilation especially in the context of organic horticulture.

Amino acid transporter mutants of Arabidopsis provides evidence that a non-mycorrhizal plant acquires organic nitrogen from agricultural soil.

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.

Plant nitrogen status and co-occurrence of organic and inorganic nitrogen sources influence root uptake by Scots pine seedlings.

Insights into how the simultaneous presence of organic and inorganic nitrogen (N) forms influences root absorption will help elucidate the relative importance of these N forms for plant nutrition in the field as well as for nursery cultivation of seedlings. Uptake of the individual N forms arginine, ammonium (NH4(+)) and nitrate (NO3(-)) was studied in Scots pine (Pinus sylvestris (L.)) seedlings supplied as single N sources and additionally in mixtures of NO3(-) and NH4(+) or NO3(-) and arginine. Scots pine seedlings displayed a strong preference for NH4(+)-N and arginine-N as compared with NO3(-)-N. Thus, NO3(-) uptake was generally low and decreased in the presence of NH4(+) in the high-concentration range (500 µM N), but not in the presence of arginine. Moreover, uptake of NO3(-) and NH4(+) was lower in seedlings displaying a high internal N status as a result of high N pre-treatment, while arginine uptake was high in seedlings with a high internal N status when previously exposed to organic N. These findings may have practical implications for commercial cultivation of conifers.

Transporters in Arabidopsis roots mediating uptake of amino acids at naturally occurring concentrations.

Recent studies of Arabidopsis have identified several transporters as being important for amino acid uptake. We used Arabidopsis plants with altered expression of lysine histidine transporter 1 (LHT1), amino acid permease 1 (AAP1) and amino acid permease 5 (AAP5) with the aim of disentangling the roles of each transporter in the uptake of different amino acids at naturally occurring concentrations (2-50 µM). LHT1 mutants displayed reduced uptake rates of L-Gln, L-Ala, L-Glu and L-Asp but not of L-Arg or L-Lys, while AAP5 mutants were affected in the uptake of L-Arg and L-Lys only. Double mutants (lht1aap5) exhibited reduced uptake of all tested amino acids. In the concentration range tested, AAP1 mutants did not display altered uptake rates for any of the studied amino acids. Expression analysis of amino acid transporter genes with important root functions revealed no major differences in the individual mutants other than for genes targeted for mutation. We conclude that LHT1 and AAP5, but not AAP1, are crucial for amino acid uptake at concentrations typically found in soils. LHT1 and AAP5 displayed complementary affinity spectra, and no redundancy with respect to gene expression was found between the two transporters, suggesting these two transporters have separate roles in amino acid uptake.