Abstract
One of the most important regulatory small molecules in plants is indole-3-acetic acid, also known as auxin. Its dynamic redistribution has an essential role in almost every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, it has not been possible to directly determine the spatial and temporal distribution of auxin at a cellular resolution. Instead it is inferred from the visualization of irreversible processes that involve the endogenous auxin-response machinery3–7; however, such a system cannot detect transient changes. Here we report a genetically encoded biosensor for the quantitative in vivo visualization of auxin distribution. The sensor is based on the Escherichia coli tryptophan repressor8, the binding pocket of which is engineered to be specific to auxin. Coupling of the auxin-binding moiety with selected fluorescent proteins enables the use of a fluorescence resonance energy transfer signal as a readout. Unlike previous systems, this sensor enables direct monitoring of the rapid uptake and clearance of auxin by individual cells and within cell compartments in planta. By responding to the graded spatial distribution along the root axis and its perturbation by transport inhibitors—as well as the rapid and reversible redistribution of endogenous auxin in response to changes in gravity vectors—our sensor enables real-time monitoring of auxin concentrations at a (sub)cellular resolution and their spatial and temporal changes during the lifespan of a plant.
Highlights
The ideal sensor for the visualization of auxin dynamics in planta should have the following features: first, physical interaction of the sensor with auxin should elicit a fluorescent signal in a reversible manner, so that changes in auxin concentration can be monitored; second, the sensitivity of the sensor should be sufficiently high to image the dynamic auxin distribution over time; third, the sensor should be targetable to different subcellular compartments—locations that are out of reach for the conventional proxies, which rely on gene expression or protein degradation; and fourth, the sensor should not contain components that are involved in plant metabolism or regulation, such that both interference with auxin responses and regulation of the sensor by the plant are avoided
The dimeric tryptophan repressor (TrpR) undergoes a conformational change upon binding TRP16,17, and fluorescent proteins fused to TrpR can relay this change, generating a fluorescence resonance energy transfer (FRET) signal as a convenient readout for in vivo measurements[18] (Fig. 1b)
Improved variants were checked for ligand specificity using a library of substances that are similar to IAA and are reportedly present in Arabidopsis (Extended Data Table 1)
Summary
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. To produce the recombinant proteins, we cloned the TrpR domain into the pET21 expression vector and generated the variants by targeted mutagenesis. This procedure was repeated with the most promising variants after each round. To obtain the individual spectra, we replaced AuxSen by mNeonGreen or Aquamarine These constructs were used for transforming plants and as a template for the unmix matrix in Fiji. Spectral imaging was performed using the QUASAR detection unit on the same system: Aquamarine was excited for FRET ratio measurement at 405 nm using 5 fluorescent channels (419–455 nm, 454–491 nm, 490–526 nm, 525–562 nm and 561–598 nm); subsequently, mNeonGreen was imaged for segmentation of the regions of interest with excitation at 488 nm and detection of 3 fluorescent channels (490–526 nm, 525–562 nm and 561–598 nm). Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this paper
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