In vivo biochemistry: applications for small molecule biosensors in plant biology

Abstract

Summary Revolutionary new technologies, namely in the areas of DNA sequencing and molecular imaging, continue to impact new discoveries in plant science and beyond. For decades we have been able to determine properties of enzymes, receptors and transporters in vitro or in heterologous systems, and more recently been able to analyze their regulation at the transcriptional level, use GFP reporters to obtain insights into cellular and subcellular localization, and measure ion and metabolite levels with unprecedented precision using mass spectrometry. However, we lack key information on location and dynamics of the substrates of enzymes, receptors and transporters, and on the regulation of these proteins in their cellular environment. Such information can now be obtained by transitioning from in vitro to in vivo biochemistry using biosensors. Genetically encoded fluorescent protein-based sensors for ion and metabolite dynamics provide highly resolved spatial and temporal information, and are complemented by sensors for pH, redox, voltage, and tension. They serve as powerful tools for identifying missing processes (e.g. glucose transport across ER membranes), components (e.g. SWEET sugar transporters for cellular sugar efflux), and signaling networks (e.g. from systematic screening of mutants that affect sugar transport or cytosolic and vacuolar pH). Combined with the knowledge of properties of enzymes and transporters and their interactions with the regulatory machinery, biosensors promise to be key diagnostic tools for systems and synthetic biology.

Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology

“…The probe concentration necessary for the analysis can be destructive and could possibly disturb the cellular equilibrium (Qin et al, 2013). Genetically encoded F€ orster resonance energy transfer (FRET) sensors provide an innovative solution to obtain a better understanding of metal homeostasis and to monitor in vivo the dynamics of metals in plants (Bermejo et al, 2011;Jones et al, 2013). FRET sensors generally consist of a substrate-specific binding domain that is flanked by two, spectrally overlapping fluorescent proteins.…”

Binary 2in1 Vectors Improve in Planta (Co)localization and Dynamic Protein Interaction Studies

“…Accordingly, two interacting proteins in close proximity, which are labeled with an appropriate donor-acceptor FP pair, will generally give rise to a strong FRET interaction, whereas proteins that are located in the same compartment but that do not interact will exhibit little or no FRET activity (Peter et al, 2014). FRET has been used not only as a tool to study protein-protein interactions (PPIs) but also as a sensing principle of small molecule sensors for the determination of a number of cellular biochemical parameters such as calcium concentrations (Miyawaki et al, 1997), changes in membrane voltage (Tsutsui et al, 2008), and hormones (Jones et al, 2013).…”

On the Promising Role of Enzyme Activity Assay in Interpreting Comparative Proteomic Data in Plants

“…Meanwhile, EAA based on ABPP should be improved to increase the number and types of enzymes that can be targeted. Additionally, a class of genetically encoded fluorescent protein‐based sensors can be targeted to subcellular compartments to monitor enzyme activity (kinases, proteases), protein conformational changes, and protein clustering …”