Figure 2.

Characterization of ABAleon2.1_Tao3s by FRET–FLIM. A, Representative pseudo-color images and data recorded by FLIM show application of ABA (20 μM)-triggered specific increases in the fluorescence lifetime of the donor τ_mT of cytosolic ABAleon2.1_Tao3 (ABAleon2.1_Tao3_cyto) and the ER ABAleon2.1_Tao3 (ABAleon2.1_Tao3_ER), but not in their corresponding donor only (mT_cyto and mT_ER) controls. For better visualization and comparisons, the color-scale bar of all the FLIM images in tobacco protoplast assays were normalized to 2.5–4.0 and the intensity bars were from 1 to 100–400 depending on the expression level of proteins in the cells. FLIM data are presented as box plots showing all data points. Significance was calculated using Student’s t test (*P < 0.05; ns, not significant). B, Representative FLIM images and data show increased τ_mT in ABAleon2.1_Tao3_cyto and ABAleon2.1_Tao3_ER upon exogenous ABA treatment at 100-nM level. FLIM data are presented as box plots showing all data points. Statistical analysis was performed using two-way ANOVA followed by Tukey’s HSD test (*P < 0.05 for overall difference between 100-nM ABA and mock treatment). C, F, Representative FLIM images of ABAleon2.1_Tao3_cyto and ABAleon2.1 (C), aABAleon2.1_Tao3_cyto and ABAleon2.1_Tao3_ER (F) in response to exogenously applied 0–100-μM ABA treatments. D, G, Shown are the plots of endogenous ABA levels in tobacco protoplasts that had been transfected with ABAleon2.1_Tao3_cyto and ABAleon2.1 (D), aABAleon2.1_Tao3_cyto, and ABAleon2.1_Tao3_ER (G) against exogenous ABA treatments. The linear equations and calculated Rsqr are shown in each plot. E, H, Shown are the τ_mT of ABAleon2.1_Tao3_cyto and ABAleon2.1 (E), aABAleon2.1_Tao3_cyto, and ABAleon2.1_Tao3_ER (H) plotted against their corresponding endogenous ABA concentrations. The ABA affinities (K′d) were calculated by fitting a four-parameter logistic curve τ = τ_min + (τ_max-τ_min)/1 + ([ABA]/K′d)ⁿ using GraphPad Prism 8.0.2. The normalized K′d (K′dN) of each ABA probe was calculated according to its linear relationship with exogenous ABA concentrations