Toward Development of Fluorescence-Quenching-Based Biosensors for Drought Stress in Plants.

Abstract

Abscisic acid (ABA) is a drought stress signaling molecule, and simple methods for detecting its levels could benefit agriculture. Here, we present 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 (PYL3). These dye-conjugated PYL3 protein form dimers in solutions without ABA and monomerize upon ABA binding. When they are in dimers, fluorescence of Cy5.5 is either nearly completely quenched by the BHQ3 or 20% quenched by another Cy5.5. Consequently, mixtures of equal amounts of the two protein conjugates were used to detect ABA in aqueous solution. As the ABA concentration increased from <1 μM to 1 mM, the 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. Kinetic modeling was used to simulate the fluorescence response from the mixture and the results generally agree with the experimentally observed trend. 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.