Triphenylphosphonium is an effective targeting moiety for plant mitochondria.

Small signalling molecules regulate a wide range of physiological and developmental processes in plants, often acting in specific spatial contexts. However, the application of such molecules - whether endogenous or synthetic - typically lacks subcellular resolution, limiting the ability to dissect their localized effects. In this study, we explored the use of triphenylphosphonium (TPP) as a mitochondrial-targeting motif in plants, using Arabidopsis thaliana and several other species as models. We synthesized and applied fluorescent TPP conjugates and analysed their distribution using confocal microscopy. To evaluate functional specificity, we designed a TPP-ciprofloxacin (CFX) conjugate and compared its activity to that of free CFX in intact plants. Fluorescent TPP conjugates consistently accumulated in mitochondria, bypassing coexisting plastids. The TPP-CFX conjugate inhibited mitochondrial DNA gyrase without affecting the chloroplast isoform, slowed plant growth, elevated mitochondrial reactive oxygen species, and induced nuclear stress-response genes, whereas free CFX perturbed both organelles. Our results establish TPP as an effective and generalizable tag for mitochondrial targeting in plant systems. This approach enables precise, organelle-specific chemical manipulation in both model and nonmodel species, offering a new tool for plant cell biology and potential applications in precision agriculture.