Aim This study was conducted to answer the question of whether clover can absorb asparagine in the presence and absence of inorganic nitrogen, as well as to determine the resulting concentration of post-uptake compounds closely involved in asparagine metabolism. Methods Clover was grown at two asparagine concentrations (10 μM and 1 mM) supplied in both the absence and presence of ammonium nitrate. Using dual-labeled 13 C 15 N-asparagine, the uptake rate was analyzed via bulk 15 N and 13 C excess and the detection of intact 13 C 15 N-asparagine in white clover. ResultsThe results from the two methods indicated greater utilization of 13 C 15 N-asparagine in the 10 μM treatment than in the 1 mM treatment. The 13 C 15 Nasparagine uptake rate was higher when 13 C 15 N-asparagine was provided alone than when it was supplemented with inorganic nitrogen. Up to nine times lower uptake rates were obtained when intact 13 C 15 N-asparagine was measured than when bulk 15 N and 13 C excess were analyzed. The labeled amino acids that are closely related to 13 C 15 N-asparagine metabolism (aspartic acid, glutamic acid and glutamine) were detected in clover roots and shoots. Conclusions Using two different methods, white clover's potential to absorb intact asparagine, even in the presence of inorganic nitrogen, was confirmed. The dual-methodology approach employed in this study demonstrates how the post-uptake metabolism can affect quantification of amino acid uptake.

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25are the most studied. In soil solution, amino acid concentrations ranges from 0.1 to 500 µM (van Hees et al. 2005) with the 26 actual availability affected by production rates, adsorption to soil particles and uptake by microorganisms and plants. 27Microorganisms largely determine the fate of amino acids due to rapid mineralization to CO2 or assimilation into cellular 28 compounds. These transformations occur within minutes (Hill et al. 2012) to hours and to assess what fraction of amino acid 29 is plant available, knowledge on kinetics of intact amino acid loss from soil is required. 30Choosing an appropriate methodology to measure dissipation rate is important. Often degradation kinetics are 67The derivatized amino acids were separated on a Waters Xbridge C18 column (2.1 mm x 150 mm). The following soil DW). The peak area of each standard was divided by the peak area of the internal 79 standard (norvaline). The normalized peak area of the standards was plotted against the standard concentration that was 80 entered in the column. A linear function was applied to the calibration curves. To determine the concentrations of the amino 81 acids and ammonium in the samples, the peak area was normalized using the peak area of norvaline and calculated using the 82 regression equations from the calibration of the standards. The minimum level at which each of the amino acids could be 83 reliably quantified was determined by the limit of detection (LOD) (Online Resource 1). Based on the background noise 84 level, the LOD was defined as the concentration of the analyte that generated a signal equal to two times the background 85 noise. Recovery experiments were performed to evaluate the efficiency and the reproducibility of the analytical method. . 86For the recovery of Asn, Asp, Gln, Gln, 4.4 g of soil was adjusted to 50% of WHC and spiked with low and high 94Recovery experiments were prepared in five replicates. Spiked soil samples were extracted and analyzed as described 102calculated from the rate constant, k, from the SFO model .

Little is known of how the deep root systems of perennial crops contribute to deeper and better resource use when intercropped with annuals in arable fields. Therefore, we aimed at measuring the capacity of perennial deep roots, alfalfa (Medicago sativa L.) and curly dock (Rumex crispus L.) to access the nutrient source located under the neighboring annuals at 1.0 and 2.5 m of soil depth. Alfalfa and curly dock were able to access the tracer-labelled source placed at a distance under the annual crop strips. As a result, the reliance on deeper soil layer for nutrient uptake under intercroppings became greater compared with sole-croppings. Combination of an annual cereal (winter rye) and a perennial legume (alfalfa) with contrasting root systems exhibited higher resource complementarity compared with intercroppings having similar root systems or absence of legumes. Our results demonstrated that the deep-rooted perennials when intercropped with annuals can induce vertical niche complementarity, especially at deeper soil layers. This was assumed to be due to the vertically stratified root activity between the crop components, however, the magnitude of the effects depended on choice of crop combinations, and on types of tracers. Future studies should include estimates such as relative yield total and land equivalent ratio to quantitatively determine the effects of resource acquisition under annual-perennial intercropping in arable fields.