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Over recent decades, many experiments have established that plants have the ability to take up intact amino acids from soil (Chapin et al., 1993; Jones & Darrah, 1993; Warren, 2006; Näsholm et al., 2009). The most common method for estimating uptake is to supply plants or soil with amino acids in which C or N are isotope-labelled (e.g. 13C and 15N, or 14C) and subsequently estimate uptake from uptake of isotope label (Näsholm et al., 1998; Näsholm & Persson, 2001; Warren, 2009b) or intact isotope-labelled amino acid molecules (Sauheitl et al., 2009a; Warren, 2012). A key aspect of studies using isotope labeling is that choices need to be made regarding which small organic N molecules are tested and which are not. In principle, the choice ought to be guided by knowledge of which forms of organic N occur in soil.
Experiments from the late 19th century until the 1970s examined uptake of many different forms of organic N (e.g. protein amino acids, nonprotein amino acids, nucleic acids, ureides, quaternary ammonium compounds, purine and pyrimidine bases) (Hutchinson & Miller, 1912; Paungfoo-Lonhienne et al., 2012), whereas in the last 20 yr the literature has focused almost exclusively on plant uptake of protein amino acids (Chapin et al., 1993; Jones & Darrah, 1993; Warren, 2006). For example, a recent review stated: ‘The concept of plant organic N nutrition relies, to a large degree, on studies of amino acids. Thus, amino acid N is in many cases used as a synonym for organic N’(Näsholm et al., 2009). Soil and/or the soil solution was long ago recognized as containing many organic N compounds in addition to protein amino acids (e.g. amino sugars, purine and pyrimidine bases, nucleic acids; e.g. see historical review: Paungfoo-Lonhienne et al., 2012), but the recent focus on protein amino acids arose because studies of the soil solution suggested amino acids were ‘the major constituent of low molecular weight dissolved organic N’ (Jones et al., 2005).
The apparent dominance of the soil solution by protein amino acids may reflect the fact that studies used targeted analytical approaches (sensu Patti et al., 2012) focused on protein amino acids plus a few common nonprotein amino acids (e.g. GABA, citrulline, ornithine) (Kielland, 1995; Turnbull et al., 1996; Andersson & Berggren, 2005; Warren, 2008; Jämtgård et al., 2010; Farrell et al., 2011a; Inselsbacher et al., 2011). Indeed, a recent broader exploration of the pool of small organic N found that, in addition to protein amino acids, the soil solution from a subalpine soil contained quaternary ammonium compounds, nonprotein amino acids, heterocyclic compounds derived from aromatic amino acids, amines, and sugar amines (Warren, 2013). Some of the single most abundant molecules were quaternary ammonium compounds, and the pool of quaternary ammonium compounds was c. 25% of the size of the pool of protein amino acids (Warren, 2013).
It is possible that quaternary ammonium compounds could be a source of N for plants, given that they can be abundant in the soil solution and plants possess transporters for quaternary ammonium compounds (Breitkreuz et al., 1999). However, at the time of writing, there was only one report of higher plants taking up quaternary ammonium compounds from soil (Audley & Tan, 1968), and with the exception of one site (Warren, 2013), little is known about the relative abundance of quaternary ammonium compounds and other compound classes that comprise the pool of small organic N. The aims of this study were to explore the abundance of quaternary ammonium compounds in a range of soils, and determine if plants can take up quaternary ammonium compounds. Capillary electrophoresis-mass spectrometry (CE-MS) was used for untargeted profiling of nonpeptide small organic N molecules (including quaternary ammonium compounds) in the soil solution of six soils. Uptake of three isotope-labelled quaternary ammonium compounds (betaine, carnitine and acetyl-carnitine) was contrasted with uptake of three protein amino acids (glycine, alanine and arginine). Uptake experiments used two ecologically divergent species: mycorrhizal wheat (Triticum aestivum L., Poaceae) a fast-growing annual grass species; and nonmycorrhizal Banksia (Banksia oblongifolia Cav., Proteaceae) a slow-growing perennial shrub that commonly occurs in nutrient-poor coastal heathlands of eastern Australia. To allow plants to express a preference for different N forms without complications from microbial competition and diffusion in soil, uptake from solution was examined with seedlings of wheat and Banksia in hydroponic solutions containing a mixture of the six forms of N at a low, field-relevant concentration of 10 μmol N l−1 each (Svennerstam et al., 2011). To examine intact uptake from soil, the six isotope-labelled N forms were injected individually into soil containing wheat plants and uptake was determined after 1 and 24 h. Intact uptake of N forms was determined from the amounts of isotope-labelled molecule that were measured within plant tissues (Persson & Nasholm, 2001; Sauheitl et al., 2009a; Warren, 2012).