Intronised fluorophores improve reporter output and biosensor expression in plants

Genetically encoded biosensors are central to plant cell biology, but low reporter signal can limit their use. Here, we developed modular intronised fluorophores to improve reporter output in transient and stable plant expression systems. Matched nuclear-localised triple mCitrine and mScarlet3 reporters were compared with and without introduced introns in Nicotiana benthamiana and Arabidopsis thaliana. Intronisation increased fluorescence, with the magnitude of enhancement depending on the fluorophore and expression context. Immunoblot analyses supported increased reporter accumulation. In Arabidopsis, intronised reporters driven by the tissue-specific SCARECROW promoter, produced stronger fluorescence signals that remained detectable farther from the root tip than non-intronised versions. Intronised versions of the abscisic acid (ABA) biosensor ABACUS2 retained ABA responsiveness and showed a lower frequency of reduced fluorescence between generations. These modular components provide a practical toolbox for improving fluorescent reporter detection and maintaining usable biosensor expression, supporting their application in plant cell biology and quantitative imaging.