The importance of each individual N compound for plant and mycorrhizal nutrition is determined by the diffusive fluxes of N through the soil to the root surface and not by soil solution concentrations (Nye, 1979; Shaver & Chapin, 1991; Leadley et al., 1997). Here we present a novel approach based on microdialysis and show that this technique can be used to assess plant-available N sources directly in the field. Our results show that the diffusive flux of N compounds in boreal forest soils is dominated by amino acids, which contributed 74–89% to the total N flux, while ammonium and nitrate amounted to only 5–15 and 5–11%, respectively (Figs 1a, S2). This dominance of amino acids was evident for all forest sites studied, including those that had been supplied with inorganic N fertilizer (Figs 2a, S2). Among the 18 individual amino acids analysed, all but three (histidine, arginine and methionine) were detected in the dialysates of 10 control (nonfertilized) soils (Fig. 3). Histidine was also found in two nonfertilized plots and in three fertilized plots (Table S2). Amino acid flux was dominated by glycine and glutamine and it was noticeable that the flux rates of both these amino acids were higher than that of ammonium, while rates of nitrate flux were only 70% of those of ammonium (Fig. 3). Compared with N flux rates, concentrations of free N estimated by water extractions of soils were dominated by ammonium, which contributed as much as 79% to the total soil solution N, while amino acids and nitrate only amounted to 11 and 10%, respectively (Figs 1b, 2b). Moreover, only nine individual amino acids were found in the water extracts (Fig. 3). Nitrogen bound to soil particles estimated by KCl extraction was also dominated by ammonium but with a larger proportion of bound amino acids contributing to the amino acid fraction and therefore to the total extracted N (Figs 2c, 3). Our results show that, in the field, plant roots and mycorrhizal fungi are exposed to almost the full range of protein amino acids (15 of 18 analysed) and may potentially use all of them for N nutrition. These findings contradict our results from soil extractions and also the current assumption that amino acids are largely bound to soil particles and, therefore, accessible for plant and mycorrhizal uptake only after exchange with other ions, such as those commonly found in root exudates. Studies of N uptake capacities of boreal forest plants (Persson & Näsholm, 2001) show that both mycorrhizal and nonmycorhizal roots may absorb the full range of protein amino acids. Moreover, recent investigations of uptake mechanisms of amino acids by plant roots and mycorrhizal fungi suggest that only a few transporters are responsible for mediating the uptake of the full range of protein amino acids, suggesting that roots and mycorrhizal hyphae cannot discriminate between uptake of individual amino acids (cf. Näsholm et al., 2009). Hence, the available information suggests that any amino acid arriving at the membrane surfaces of roots or mycorrhizal hyphae will be absorbed at rates comparable to that of ammonium and appreciably higher than that of nitrate. Further, our study indicates that in boreal forests, amino acids dominate soil N supply. This result is in line with studies of Arctic (Kielland, 1994; Kielland et al., 2007) and boreal ecosystems (Nordin et al., 2001) but contrasts with several previous studies suggesting that inorganic N, particularly in the form of ammonium, is the major source of N available to plants and mycorrhizal fungi (Likens et al., 1969; Robertsson, 1982; Kronzucker et al., 1997; Rothstein, 2009).
Figure 2. Nitrogen (N) diffusive fluxes estimated by microdialysis sampling (a), free soil N concentrations estimated by water extraction (b), and free and exchangeable soil N concentrations (c) in boreal forest sites receiving different amounts of N fertilizer (green bars, 0 kg N; blue bars, 6 kg N; red bars, 50 kg N). Bars represent means ± SE of three forest sites (n for dialysis = 60, n for extracts = 27). AA tot, total amino acids.
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Eutrophication of terrestrial ecosystems, resulting from fertilizer additions or anthropogenic N deposition, occurs mainly through input of mineral N forms (Vitousek et al., 1997; Galloway et al., 2008). The forests in our study are located in an area that receives very low background deposition of N (2 kg N ha−1 yr−1) and we expected that in forests exposed to higher rates of mineral N input, plant-available N would shift towards ammonium and nitrate and that organic N would constitute a smaller proportion of this pool. Diffusive fluxes of soil N in nonfertilized plots in natural, N-limited forests were 8.3, 1.5 and 0.9 nmol N cm−2 h−1 for amino acid N, ammonium and nitrate, respectively (Fig. 2, Table S2). Surprisingly, long-term fertilization with NH4NO3 resulted in higher flux rates of amino acids, increasing from 8.3 nmol N cm−2 h−1 in unfertilized plots to 10.1 nmol N cm−2 h−1 at an addition rate of 6 kg N ha−1 yr−1 and 14.8 nmol N cm−2 h−1 at 50 kg N ha−1 yr−1, while no such trend was observed for the two inorganic N forms (Fig. 2, Table S2). The fractions of N diffusing across the dialysis membrane that were accounted for by amino acids were, for the three N concentrations, 77.3, 81.3 and 83.6%, illustrating the potential dominance of amino acids as N sources for plants and mycorrhizal fungi in low-, medium- and high-N environments. This unexpected effect of inorganic N fertilizer application on supply rates of soil amino acids, but not on inorganic N, suggests that added N had a major effect on the overall N cycling in the forest soil by increasing the activity of soil microbes and roots. The fact that inorganic N remained at similar concentrations in all N treatments suggests that the later steps in the N mineralization pathway, such as ammonification and nitrification, may not limit production of available N (Schimel & Bennett, 2004; Kielland et al., 2007; Wanek et al., 2010) and instead emphasizes the importance of depolymerization of proteins to peptides and free amino acids as a major bottleneck in forest N cycling. By contrast, the effects of N fertilization on amino acid fluxes revealed by our in situ dialysis measurements were not detected after soils were sieved, homogenized and extracted in the laboratory according to standard techniques for characterizing soil N concentrations (Fig. 2, Tables S3, S4).
Plants may acquire N from a wide range of N compounds (Näsholm et al., 2009), and, among the organic forms of N, amino acids are of special importance because of their relatively small size and rapid uptake rates. Here we show that induced diffusive flux of amino acids dominate over ammonium and nitrate in boreal forest soils, suggesting that such N forms play a critical role in the N nutrition of plants in this biome. Induced diffusive flux of amino acids was also responsive to long-term N fertilization, while no such response could be detected for either of the inorganic N sources, suggesting that amino acids may be primary N sources also in soils of higher fertility. Plant N acquisition, in particular in N-poor soils, depends on diffusion driven by the active uptake by plant roots and mycorrhizal hyphae. This fact, together with the apparent problem of using destructive techniques for studying compounds such as amino acids that exhibit high turnover rates in soils (Jones & Kielland, 2002), points to a need for new approaches in studies of plant–soil interactions, one of which is presented here.