Abstract

Summary Activity‐based sensing probes are powerful tools for monitoring enzymatic activities in complex biological samples such as cellular and live animals; however, their application in plants remains challenging. Herein, fourteen activity‐based fluorescent probes were assayed against Arabidopsis O ‐methyltransferases ( At OMTs). One probe, 3‐BTD, displayed a high selectivity, reactivity, and fluorescence response toward At OMTs especially the isoform At CCoAOMT. We further characterized the features of this probe and explored whether it could be used to detect OMT activities in living plant cells. Our results show that 3‐BTD can be used to visualize OMT activity in Arabidopsis , and no fluorescent signal was observed in the comt / ccoaomt double mutant, indicating that it has good specificity. Interestingly, in contrast to the observation that At CCoAOMT‐YFP accumulated in both cytoplasm and nucleus, OMT enzymatic activity tracked by 3‐BTD probe was found only in the cytoplasm. This underscores the importance of activity‐based sensing in studying protein function. Moreover, 3‐BTD can be successfully applied in OMT visualization of different plants. This study indicates that 3‐BTD can serve as a potential probe for in situ monitoring the real activity of OMT in multiple plants and provides a strategy for visualizing the activity of other enzymes in plants.