Multiparameter in vivo imaging in plants using genetically encoded fluorescent indicator multiplexing

Biological processes are highly dynamic, and during plant growth, development, and environmental interactions, they occur and influence each other on diverse spatiotemporal scales. Understanding plant physiology on an organismic scale requires analyzing biological processes from various perspectives, down to the cellular and molecular levels. Ideally, such analyses should be conducted on intact and living plant tissues. Fluorescent protein (FP)-based in vivo biosensing using genetically encoded fluorescent indicators (GEFIs) is a state-of-the-art methodology for directly monitoring cellular ion, redox, sugar, hormone, ATP and phosphatidic acid dynamics, and protein kinase activities in plants. The steadily growing number of diverse but technically compatible genetically encoded biosensors, the development of dual-reporting indicators, and recent achievements in plate-reader-based analyses now allow for GEFI multiplexing: the simultaneous recording of multiple GEFIs in a single experiment. This in turn enables in vivo multiparameter analyses: the simultaneous recording of various biological processes in living organisms. Here, we provide an update on currently established direct FP-based biosensors in plants, discuss their functional principles, and highlight important biological findings accomplished by employing various approaches of GEFI-based multiplexing. We also discuss challenges and provide advice for FP-based biosensor analyses in plants.