Imaging of Multiple Plant Hormones in Roots of Rice (Oryza sativa) Using Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

Plant hormones can act in synergistic and antagonistic ways in response to biotic and abiotic stresses and in plant growth and development. Thus, a technique is needed to simultaneously determine the distributions and concentrations of several plant hormones. Previously, we reported that localizations of two plant hormones [cytokinin (CK) and abscisic acid (ABA)] can be simultaneously visualized in a plant tissue using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). In MALDI-MS, however, self-ionization of an organic matrix occasionally interferes with ionizations of small molecules (<500 m/z) including most plant hormones. Another technique, nanoparticle-assisted laser desorption/ionization (Nano-PALDI), can avoid matrix self-ionization using nanoparticles to assist the ionization of analytes. Here, we compared the ionization efficiencies of common plant hormones by MALDI-MS and Nano-PALDI-MS. For the comparison, we prepared a standard mix of seven plant hormones [ABA, auxin (IAA), brassinosteroid (Br), two CKs (trans-zeatin, tZ, and 6-(gamma,gamma-dimethylallylamino) purine, iP), jasmonic acid, and salicylic acid (SA)], an ethylene precursor (1-aminocyclopropane-1-carboxylic acid, ACC), and a heavy hydrogen-labeled ABA (D-6-ABA). Basic MALDI-MS detected all compounds except IAA, Br, and D-6-ABA, while Nano-PALDI-MS detected all nine compounds. By Nano-PALDI-MS imaging (MSI), each of the abovementioned hormones and ACC were also detected in root cross sections of rice which were incubated in the hormone mix for 2 h. In the elongation zone of untreated roots, Nano-PALDI-MSI revealed high levels of ABA and CKs in the outer part of roots and much lower levels in the stele, but Br, SA, and ACC were broadly distributed in the cross section. IAA seemed to be distributed in the epidermis, cortex, and stele. Multiple-hormone imaging using Nano-PALDI-MS has great potential for investigating the roles of hormone signaling in crop development and stress responses.