A suite of fluorescent timers for visualizing protein spatiotemporal dynamics in plants.

Fluorescent timers (FTs), including tandem FTs (tdFTs), enable real-time visualization of precise protein localization and turnover within living cells, an essential property for elucidating protein function. In plants, application of FT has been limited largely because of the mismatch between FT maturation kinetics and the slow growth rate of plant cells. Here, we systematically evaluated multiple FT configurations to identify tdFTs optimized for plant systems. Using Arabidopsis thaliana, we tested a range of fluorescent proteins (FPs) differing in maturation times. Functional tdFT expression was confirmed in diverse organelles. Selected tdFT configurations were developed to track protein biogenesis/turnover in autoimmunity, protein transport in abiotic stress, and polarized protein translocation in determination of cell fate. When the large size of tdFT interfered with protein mobility, we developed a bipartite FT, separately tagging proteins with different FPs. This resource provides a framework for selecting FTs to dissect dynamic protein behaviors in plant cells.