Mass spectrometry‐based quantification and spatial localization of small organic acid exudates in plant roots under phosphorus deficiency and aluminum toxicity

Abstract

Low-molecular-weight organic acid (OA) 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 OA 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 inorganic ortho-phosphate (Pi) deficiency and aluminum 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 ultra-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (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 min per sample. We also employed targeted (MS/MS) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to detect the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods, we studied OA 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 analyzing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.

Label-free functional analysis of root-associated microbes with dynamic quantitative oblique back-illumination microscopy

“…This level of increased dynamics suggests higher levels of plant-secreted carbon compounds in the root cap attracting bacteria and encouraging replication. Such a possibility is consistent with reports of higher production of arabinogalactan-proteins and certain organic acids at root tips 38 , 39 , which can serve as carbon and energy sources for the bacteria. Phasor plots from the colonized roots can be seen in Supplemental Section S3 .…”

Seeing plants as never before

“…Stress‐induced changes in metabolite localization is not exclusive to lipids. Arabidopsis roots under altered phosphate conditions showed differences in the exudation of organic acids citrate and malate (Gomez‐Zepeda et al ., 2021). Finally, a comparative MSI (and NMR) study showed different drought‐tolerance strategies utilized within roots of three tropical plant species ( Piper sp., Hibiscus rosa sinensis , Clitoria fairchildiana ), with heterogeneous spatial patterns in a range of metabolites associated with structural, redox, and microbial defense (Honeker et al ., 2022).…”

Metabolomics of plant root exudates: From sample preparation to data analysis

“…However, further optimization in sample preparation is indispensable for better spectral quality ( Dias et al., 2016 ). Further, MSI-based techniques were utilized to directly depict the spatial distribution of metabolites in the rhizosphere of Zea mays L. plants, Brachypodium distachyon roots, Arabidopsis seedlings, Asparagus roots, tomato roots and Marchantia polymorpha gemmalings ( Sasse et al, 2020 ; Korenblum et al., 2020 ; Döll et al., 2021 ; Gomez-Zepeda et al., 2021 ; Lohse et al., 2021 ). Additionally, LSC-MS allows for the detection of hundreds of plant-specific metabolites acquired from a single plant cell, discriminating them from those produced by soil microbes ( Masuda et al., 2018 ; Taylor et al., 2021 ).…”