Visualizing Cellular Gibberellin Levels Using The Nisgps1 Forster Resonance Energy Transfer (Fret) Biosensor

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first Forster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nIsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nIsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A(4) (GA(4)) treatments, and over a treatment time-course. We also provide methods to analyze nIsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nIsGPS1 to quantify gibberellin levels.