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

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.

Functional Characterization of the Lin28/let-7 Circuit During Forelimb Regeneration in Ambystoma mexicanum and Its Influence on Metabolic Reprogramming.

The axolotl (Ambystoma mexicanum) is a caudate amphibian, which has an extraordinary ability to restore a wide variety of damaged structures by a process denominated epimorphosis. While the origin and potentiality of progenitor cells that take part during epimorphic regeneration are known to some extent, the metabolic changes experienced and their associated implications, remain unexplored. However, a circuit with a potential role as a modulator of cellular metabolism along regeneration is that formed by Lin28/let-7. In this study, we report two Lin28 paralogs and eight mature let-7 microRNAs encoded in the axolotl genome. Particularly, in the proliferative blastema stage amxLin28B is more abundant in the nuclei of blastemal cells, while the microRNAs amx-let-7c and amx-let-7a are most downregulated. Functional inhibition of Lin28 factors increase the levels of most mature let-7 microRNAs, consistent with an increment of intermediary metabolites of the Krebs cycle, and phenotypic alterations in the outgrowth of the blastema. In summary, we describe the primary components of the Lin28/let-7 circuit and their function during axolotl regeneration, acting upstream of metabolic reprogramming events.