Pyridinium Derivatives as Novel Fluorescent Probes for Xylem Staining in Plant Tissues

Vascular plants (tracheophytes) rely on specialized tissues, xylem and phloem, for structural support, nutrient transport, and physiological adaptation. Understanding the structural and functional properties of these vascular tissues is essential for elucidating plant development and stress responses. Traditional staining methods, which utilize both fluorescent and non-fluorescent dyes, have been employed to visualize xylem and phloem; however, their limited specificity and sensitivity restrict detailed analysis. To overcome these limitations, we synthesized novel cationic pyridinium derivatives with enhanced sensitivity-enabled by intramolecular charge transfer-and high specificity for xylem tissues across diverse plant species. Comparative staining with traditional dyes such as propidium iodide, berberine, basic fuchsin, and rhodamine confirmed the superior sensitivity of these derivatives, while tissue-specific assays demonstrated strong xylem selectivity with minimal background noise across diverse plant species. Furthermore, staining of the Arabidopsis eskimo1 mutant with a collapsed xylem vessel phenotype indicated the utility of these derivatives in developmental biology studies. Our study presents a faster, specific, and highly sensitive staining method using pyridinium derivatives, offering a powerful tool for advancing research on xylem biology.