Other methods are available for the detection of bioavailable ABA, spanning from mass spectrometry 6, 7 to antibody-based immunoassays such as commercially available ELISA 8, 9, to electrochemical 10 and optical sensors not based on FRET 11, 12. 5 These pioneering works suggest that it is possible to develop sensitive optical sensors to detect ABA in plants. Other similar methods have been developed. 4 Upon ABA binding, the phosphatase binds to the PYL to enable FRET, causing decrease of fluorescence in one spectral region and increase in another. One report demonstrated the quantification of μM concentrations of ABA in plants by obtaining the ratio of fluorescence intensities in two spectral regions from two fluorophores, one attached to a PYL and the other to a phosphatase. PYL-based ABA sensors have been investigated by designing recombinant proteins that incorporate fluorescent domains to enable optical sensing using principles such as Förster resonance energy transfer (FRET). The PYL therefore present viable candidates to engineer a biosensor. These sensors would allow plants to report to farmers or automated irrigation systems to obtain point-of-mitigation. Thus, an engineered interface to in vivo ABA signaling utilizing endogenous components as biosensors represents the best ready-to-use mechanism to detect micromolar (μM) level ABA in drought-stressed plants and could enable real time human-mediated mitigation of drought. Throughout evolution, this response to drought stress has been finely tuned, making it challenging for humans to conveniently detect subtle, yet physiologically relevant changes in ABA concentration without the use of these naturally designed reporters. 2, 3 In the absence of ABA, the dimeric receptors are autoinhibited, enabling PP2C phosphatases to bind to SnRK2 kinases, rendering them inactive. Upon ABA-binding, the dimers dissociate to their ABA-bound monomeric forms, which then regulate PP2C phosphatases and activate SnRK2 kinases that direct many downstream signaling pathways including the control of stomata aperture. 1 The ABA signaling network in plants involves a class of water-soluble ABA receptor proteins (PYR/PYL/RCAR), which form dimers in absence of ABA. Facing drought or other stresses, for example, plants synthesize and respond to a terpenoid hormone called abscisic acid (ABA), which is involved in seed germination, seedling growth, regulation of stomatal aperture, flowering and response to pathogens. In response to environmental stresses, plants can adjust growth and development using phytohormones. This work demonstrates that fluorescence measurements of a single dissociation reaction in one spectral region are adequate to assess the ABA concentration of a solution. Kinetic modeling was used to simulate the fluorescence response from the mixture and the results generally agree with the experimentally observed trend. As the ABA concentration increased from less than one μM to one mM, intensity of fluorescence detected at around 680 nm from the mixture was more than doubled as a result of ABA induced monomerization, which leads to halt of quenching and recovery of fluorescence of Cy5.5 in monomers. Consequently, mixtures of equal amounts of the two protein conjugates were used to detect ABA in aqueous solution. When in dimers, fluorescence of Cy5.5 is either nearly completely quenched by the BHQ3 or 20% quenched by another Cy5.5. These dye-conjugated PY元 form dimers in solutions without ABA and monomerize upon ABA binding.
Here, we present as proof-of-concept detection for ABA in aqueous solutions by the use of a mixture of Cyanine 5.5 (Cy5.5) fluorophore- and BHQ3 quencher-conjugated endogenous ABA receptor pyrabactin resistance 1 like proteins (PY元). Abscisic acid (ABA) is a drought stress signaling molecule and simple methods for detecting its levels could benefit agriculture.
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