Relative Protein Lifetime Measurement in Plants Using Tandem Fluorescent Protein Timers

Targeted protein degradation plays a wide range of important roles in plant growth and development, but analyzing protein turnover in vivo is technically challenging. Until recently, there has been no straightforward methodology for quantifying protein dynamics at subcellular resolution during cellular transitions in plants. A tandem fluorescent protein timer (tFT) is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which allows estimation of relative protein age from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we describe approaches to use this technology to report relative protein lifetime in both transient and stable plant transformation systems. tFTs enable in vivo, real-time protein lifetime assessment within subcellular compartments and across tissues, permitting the analysis of protein degradation dynamics in response to stresses or developmental cues and in different genetic backgrounds.