Mass spectrometry-based quantification and spatial location of small organic acids exudate in plant roots under phosphorous deficiency and aluminium toxicity

Low-molecular-weight organic acids (OAs) extrusion by plant roots is critical for plant nutrition, tolerance to cations toxicity, and plant-microbe interactions. Therefore, methodologies for the rapid and precise quantification of OAs are necessary to be incorporated in the analysis of roots and their exudates. The spatial location of root exudates is also important to understand the molecular mechanisms directing OAs production and release into the rhizosphere. Here, we report the development of two complementary methodologies for OA determination, which were employed to evaluate the effect of ortho-phosphate (Pi) deficiency and aluminium toxicity on OA excretion by Arabidopsis roots. OA exudation by roots is considered a core response to different types of abiotic stress and for the interaction of roots with soil microbes, and for decades has been a target trait to produce plant varieties with increased capacities of Pi uptake and Al tolerance. Using targeted UPLC-HRMS/MS, we achieved the quantification of six OAs in root exudates at sub-micromolar detection limits with an analysis time of less than 5 minutes per sample. We also employed targeted (MS/MS) MALDI-MS imaging to detect the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods, we studied OAs exudation in response to Al toxicity and Pi deficiency in Arabidopsis seedlings overexpressing genes involved in OA excretion. Finally, we show the transferability of the MALDI-MSI method by analysing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.