Triphenylphosphonium is an effective targeting moiety for plants mitochondria

Small signaling molecule regulates key physiological processes in plants, often in a spatially distinct manner. However, current methods for applying small-molecules, endogenous or synthetic, in plants research lack spatial precision, limiting the ability to study and utilize their localized effects. Here, we validate triphenylphosphonium (TPP) as a mitochondrial targeting motif in plants. Using fluorescently labeled TPP conjugates in Arabidopsis thaliana, we demonstrate mitochondria-specific accumulation, even in the presence of plastids. This precise localization enables detailed imaging of mitochondria and mitochondrial DNA in living plants. We further exploit this targeting ability by developing a TPP-ciprofloxacin conjugate to selectively inhibit mitochondrial DNA gyrase, an enzyme involved in organellar DNA replication. Unlike free ciprofloxacin, which disrupts both mitochondrial and chloroplast DNA gyrase activity, the TPP-conjugate specifically targets mitochondrial gyrase, leading to slower plant growth without affecting chloroplast function. This targeted inhibition triggers a mitochondrial retrograde response, characterized by increased reactive oxygen species levels and the upregulation of stress-response genes in the nucleus. Our findings establish TPP as a reliable tool for mitochondrial targeting in plants and open avenues for both fundamental research and agricultural applications. By enabling organelle-specific manipulation in species not amenable to genetic engineering, TPP-based strategies have potential for advancing plant biology and precision agriculture.