Figure 1

Biochemical characterization of Arabidopsis ABA receptors by a wheat cell-free system. (a) Principle of receptor-PP2CA interaction analysis by the AlphaScreen. Biotinylated-ABA receptor binds to streptavidin on the donor beads with an extremely specific and high affinity. The protein A-coated acceptor beads are combined with FLAG-PP2CA through the anti-FLAG antibody. ABA receptor-PP2CA complex forms a large complex with two kinds of beads through the antibody and streptavidin. After illumination at 680 nm, the donor beads convert ambient oxygen to singlet oxygen (¹O₂), and singlet oxygen is transferred across to activate the acceptor beads and subsequently emits light at 520–620 nm. (b) Interaction analysis of biotinylated PYR and FLAG-ABI1. Recombinant biotinylated PYR and FLAG-ABI1 were incubated in the absence (−) or presence (+) of 10 μM ABA. FLAG-PTP1 was used as the negative control. (c) ABA-dependent interaction analysis of biotinylated PYR and FLAG-ABI1. The final concentration (M) of ABA is indicated in the graph. Relative AlphaScreen signal (AS) was expressed as a relative value with the signal of DMSO as one. (d) Interaction analysis of 14 biotinylated ABA receptors and FLAG-ABI1. Relative AlphaScreen signals were expressed as a relative value with the PYR1-ABI1 interaction signal value in the presence of 100 μM ABA taken as 100%. (e) Interaction analysis of 14 biotinylated ABA receptors and FLAG-ABI1, ABI2, HAB1, HAI1, HAI2, AHG1, and AHG3 in the absence (−) or presence (+) of 100 μM ABA. FLAG-DHFR was used as the negative control. Relative AlphaScreen signal was expressed as a relative value with the signal of FLAG-DHFR as one. (f) A heat map of the protein–protein interaction AlphaScreen signals between 14 biotinylated ABA receptors and 14 AGIA-ABA receptors. Recombinant ABA receptors were incubated in the absence (left) or presence (middle) of 100 μM ABA. Biotinylated DHFR was used as the negative control. Relative AlphaScreen signal was expressed as a relative value with the signal of biotinylated DHFR as one. The relative change in interaction signal induced by ABA treatment is expressed as log₂ compared with the signal value of DMSO (right). Error bars represent standard deviations (n = 3).