Advantages of compound‐specific stable isotope measurements over bulk measurements in studies on plant uptake of intact amino acids

Abstract

Increasing interest in the ability of plants to take up amino acids has given rise to questions on the accuracy of the commonly used bulk method to measure and calculate amino acid uptake. This method uses bulk measurements of 13C and 15N enrichment in plant tissues after application of dual-labelled amino acids but some authors have recommended the use of compound-specific stable isotope (CSI) analysis of the plants' amino acids instead. However, there has never been a direct evaluation of both methods. We conducted a field study applying dual-labelled (13C, 15N) amino acids (glycine, valine, tyrosine and lysine) to soil of a Plantago lanceolata monoculture. Root and shoot samples were collected 24 h after label application and the isotope composition of the plant tissues was investigated using bulk and CSI measurements. Enrichment of 13C in the case of CSI measurements was limited to the applied amino acids, showing that no additional 13C had been incorporated into the plants' amino acid pool via the uptake of tracer-derived C-fragments. Compared with this rather conservative indicator of amino acid uptake, the 13C enrichment of bulk measurements was 8, 5, 1.6 and 6 times higher for fine roots, storage roots, shoot and the whole plant, respectively. These findings show that the additional uptake of tracer-derived C-fragments will result in a considerable overestimation of amino acid uptake in the case of bulk measurements. We therefore highly recommend the use of CSI measurements for future amino acid uptake studies due to their higher accuracy.

Simultaneous efflux and uptake of metabolites by roots of wheat

“…6). The presence of U-13 C isotopologues within roots is arguably more conservative and definitive evidence for uptake of metabolites because with this approach one may be certain that the molecules were taken up intact (Persson and Nasholm 2001;Sauheitl et al 2009;Warren 2012). The major drawback of examining amounts of U-13 C isotopologues within roots is that plants were in the 13 C-metabolite solution for four hours during which time substantial post-uptake metabolism (and attendant scrambling of 13 C atoms) probably decreased the amounts of U-13 C isotopologue present within roots (Warren 2012).…”

“…Uptake was estimated in two ways. First, from the decrease in concentration of the U-13 C isotopologue in the uptake solution; and second, from the amounts of U-13 C isotopologue that appeared within roots (Persson and Nasholm 2001;Sauheitl et al 2009;Warren 2012). Analysis of the uptake solution also permitted estimation of rates of efflux based on the appearance of U-12 C isotopologues.…”

Direct acquisition of organic N by white clover even in the presence of inorganic N

“…One criticism of the literature regarding amino acid absorption is that some of the studies have measured recovery of 15 N in the target tissue after application of 15 N-labeled amino acids (Lesuffleur and J-B 2010a;Lesuffleur et al 2007) or analyzed bulk 13 C and 15 N enrichments after exposure to 15 N, 13 C-dual labeled amino acid (Näsholm et al 1998;Näsholm et al 2000). The main limitation of the bulk 15 N and/or 13 C measurements is that it cannot separate the acquisition of intact molecules from the acquisition of molecules that were transformed prior to uptake, which may lead to either uptake overestimation (Rasmussen et al 2010;Sauheitl et al 2009a) or underestimation due to respiration loss of 13 C (Warren 2012). Clear evidence for uptake is provided by the detection of intact labeled amino acids in plant material determined by e.g., gas or liquid chromatography mass spectrometry (Nordin et al 2004;Öhlund and Näsholm 2001;Persson et al 2003;Persson and Näsholm 2001;Warren 2012).…”

Warming alters competition for organic and inorganic nitrogen between co-existing grassland plant species

“…In other words, residual carbon, including 13 C, might have been respired to a higher extent when applied as peptide than as amino acid, resulting in a smaller 13 C 15 N ratio. To reduce such uncertainties about direct uptake of labelled isotopes in future experiments, the application of other techniques might be helpful, such as compound-specific stable isotope measurements (Sauheitl et al 2009a), positionspecific labeling (Apostel et al 2013) and the use of 14 C-labelled isotopes ). However, all available techniques are subject to some caveats and assumptions.…”