A Protocol for Activated Bioorthogonal Fluorescence Labeling and Imaging of 4‐Hydroxyphenylpyruvate Dioxygenase in Plants

Abstract 4‐Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene‐linked dibenzyl‐cyclooctyne undergoes strain‐promoted azide‐alkyne cycloaddition with an azide‐containing HPPD ligand. The activation‐based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana , and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD‐related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.