Background: Biofortification of staple crops with essential micronutrients relies on the efficient, long distance transport of nutrients to the developing seed. The main route of this transport in common wheat (Triticum aestivum) is via the phloem, but due to the reactive nature of some essential micronutrients (specifically Fe and Zn), they need to form ligands with metabolites for transport within the phloem. Current methods available in collecting phloem exudate allows for small volumes (μL or nL) to be collected which limits the breadth of metabolite analysis. We present a technical advance in the measurement of 79 metabolites in as little as 19.5 nL of phloem exudate. This was achieved by using mass spectrometry based, metabolomic techniques.
Results: Using gas chromatography-mass spectrometry (GC-MS), 79 metabolites were detected in wheat phloem. Of these, 53 were identified with respect to their chemistry and 26 were classified as unknowns. Using the ratio of ion area for each metabolite to the total ion area for all metabolites, 39 showed significant changes in metabolite profile with a change in wheat reproductive maturity, from 8-12 to 17-21 days after anthesis. Of these, 21 were shown to increase and 18 decreased as the plant matured. An amine group derivitisation method coupled with liquid chromatography MS (LC-MS) based metabolomics was able to quantify 26 metabolites and semi-quantitative data was available for a further 3 metabolites.
Conclusions: This study demonstrates that it is possible to determine metabolite profiles from extremely small volumes of phloem exudate and that this method can be used to determine variability within the metabolite profile of phloem that has occurred with changes in maturity. This is also believed to be the first report of the presence of the important metal complexing metabolite, nicotianamine in the phloem of wheat.
Keywords: Aphid stylectomy; Exudate; GC-MS; Grain loading; LC-MS; Metabolomics; Method development; Phloem; Wheat.