Enhancement of in situ detection and imaging of phytohormones in plant tissues by MALDI‐MSI using 2,4‐dihydroxy‐5‐nitrobenzoic acid as a novel matrix

Summary Phytohormones possess unique chemical structures, and their physiological effects are regulated through intricate interactions or crosstalk among multiple phytohormones. MALDI‐MSI enables the simultaneous detection and imaging of multiple hormones. However, its application for tracing phytohormones is currently restricted by low abundance of hormone in plant and suboptimal matrix selection. 2,4‐Dihydroxy‐5‐nitrobenzoic acid (DHNBA) was reported as a new MALDI matrix for the enhanced detection and imaging of multiple phytohormones in plant tissues. DHNBA demonstrates remarkable sensitivity improvement when compared to the commonly used matrix, 2,5‐dihydroxybenzoic acid (DHB), in the detection of isoprenoid cytokinins ( trans ‐zeatin ( t Z), dihy‐drozeatin (DHZ), meta ‐topolin ( m T), and N 6 ‐(Δ 2 ‐isopentenyl) adenine (iP)), jasmonic acid (JA), abscisic acid (ABA), and 1‐aminocyclo‐propane‐1‐carboxylic acid (ACC) standards. The distinctive properties of DHNBA (i.e. robust UV absorption, uniform matrix deposition, negligible background interference, and high ionization efficiency of phytohormones) make it as an ideal matrix for enhanced detection and imaging of phytohormones, including t Z, DHZ, ABA, indole‐3‐acetic acid (IAA), and ACC, by MALDI‐MSI in various plant tissues, for example germinating seeds, primary/lateral roots, and nodules. Employing DHNBA significantly enhances our capability to concurrently track complex phytohormone biosynthesis pathways while providing precise differentiation of the specific roles played by individual phytohormones within the same category. This will propel forward the comprehensive exploration of phytohormonal functions in plant science.