Focus on biosensors: Looking through the lens of quantitative biology

Abstract

In recent years, plant biologists interested in quantifying molecules and molecular events in vivo have started to complement reporter systems with genetically encoded fluorescent biosensors (GEFBs) that directly sense an analyte. Such biosensors can allow measurements at the level of individual cells and over time. This information is proving valuable to mathematical modellers interested in representing biological phenomena in silico, because improved measurements can guide improved model construction and model parametrisation. Advances in synthetic biology have accelerated the pace of biosensor development, and the simultaneous expression of spectrally compatible biosensors now allows quantification of multiple nodes in signalling networks. For biosensors that directly respond to stimuli, targeting to specific cellular compartments allows the observation of differential accumulation of analytes in distinct organelles, bringing insights to reactive oxygen species/calcium signalling and photosynthesis research. In conjunction with improved image analysis methods, advances in biosensor imaging can help close the loop between experimentation and mathematical modelling.

Next-generation ABACUS biosensors reveal cellular ABA dynamics driving root growth at low aerial humidity

“…The sites of ABA biosynthesis, metabolism and translocation are the subject of intensive research, but progress has been hampered by limitations in tools to quantify accumulation and depletion of ABA on a tissue/cellular scale where regulatory decisions controlling ABA dynamics are made 9,10 . The availability of sensitive reporters, particularly Förster Resonance Energy Transfer (FRET) biosensors, for hormones, second messengers and metabolism are revolutionizing plant development, signalling and photosynthesis research 11 . Such biosensors are powerful tools to quantify metabolites in vivo at high spatiotemporal resolution 11 , including phytohormones under changing environmental conditions 12–16 .…”

“…This produced our highest ratio change biosensor that has ABA sensitivity suitable for in planta studies, which we named ABACUS2-400n (KD (ABA): Improved promoter/terminator combinations allowed us to express ABACUS2 sensors with nuclear localization signals (nls) in wildtype Arabidopsis thaliana (Col-0), overcoming our previously severe ABACUS1 silencing problems 14 . Nuclear localization allows easy discrimination of the fluorescence of neighbouring cells and the exclusion of non-nuclear background and auto-fluorescence during image processing 11 . To accelerate this image processing, we developed a comprehensive image analysis toolset to quickly analyse confocal stacks in 3D/4D, allowing us to robustly quantify and visualize nuclear emission ratios within moments (See supplemental methods and 26 ).…”

“…quantify metabolites in vivo at high spatiotemporal resolution 11 , including phytohormones under changing environmental conditions [12][13][14][15][16] . Direct ABA FRET biosensors that do not require additional signalling components have broad application potential beyond ABA quantification in plant cells and sub-cellular compartments.…”

“…We selected the first mutation, PYL1 R143S, to alter water mediated PYL1-ABA-ABI1aid interactions. This produced our highest ratio change biosensor that has ABA sensitivity suitable for in planta studies, which we named ABACUS2-400n (KD (ABA): 11 . To accelerate this image processing, we developed a comprehensive image analysis toolset to quickly analyse confocal stacks in 3D/4D, allowing us to robustly quantify and visualize nuclear emission ratios within moments (See supplemental methods and 26 ).…”

Encoding, transmission, decoding, and specificity of calcium signals in plants

“…Some related challenges in relation to interpreting calcium images have been described ( Vaz Martins and Livina, 2019 ). Excellent reviews on the latest imaging advances have recently been published ( Clark et al , 2020 ; Grenzi et al , 2021 ; Rowe and Jones, 2021 ; Sadoine et al , 2021 ).…”