Figure 6.
ABA Is Important for ABAR–WRKY40 Interaction, and the Promoting Effect of ABA on ABAR–WRKY40 Interaction Requires Function of ABAR.
(A) Interaction of ABAR and WRKY40 in the ABA-deficient mutant aba2 cells. ABAR-YFPN/WRKY40-YFPC –ABA, BiFC in the absence of exogenous ABA treatment; ABAR-YFPN/WRKY40-YFPC +ABA, BiFC with 2 μM ABA treatment 2 h before observation. The protoplasts transformed with the construct pair ABAR-YFPN/YFPC with 0 μM (– ABA) or 2 μM ABA (+ ABA) treatment were taken as negative controls. Images were taken with identical parameters to allow comparison of fluorescence intensities.
(B) Immunoprecipitation (IP) assays in the aba2 plants show that ABA is required for the ABAR–WRKY40 interaction. (±)ABA at 0.1 μM was added into the isolated total protein for an incubation of 4 h at 4°C before the IP assays were conducted. (a) IP with anti-ABAR serum and immunoblotting (Blot) with both sera, and (b) IP with anti-WRKY40 serum and Blot with both sera. The symbols – and + indicate ABA-free and ABA treatment, respectively. Protein amounts were evaluated by scanning the protein bands, and relative band intensities, normalized relative to the intensity with the value from the sample of the ABA-free treatment (as 100%), are indicated by numbers below the bands. The experiments were repeated three times with the similar results.
(C) Promoting effect of ABA on ABAR–WRKY40 interaction requires ABAR function. Tobacco leaves were transformed with the construct pairs CLuc-abar/WRKY40-NLuc and CLuc-ABAR/WRKY40-NLuc (a), abar-NLuc/CLuc-WRKY40 and ABAR-NLuc/CLuc-WRKY40 (b), or CLuc-abar/NLuc and CLuc-ABAR/NLuc ([c]; as a negative control). The leaves were observed for fluorescence imaging 6 h after the (±)ABA infiltration (0 μM, indicated by −ABA, or 80 μM, indicated by +ABA) by a needleless syringe. The term abar denotes the ABAR gene harboring the cch mutation. Top panels in (a) to (c) show fluorescent images (LUC); middle panels show corresponding locations of transformation in the leaf with the given construct pairs. The bottom panels show the corresponding quantitative data (each value is the mean ± se with five independent determinations): top and bottom columns in (a), CLuc-abar/WRKY40-NLuc and CLuc-ABAR/WRKY40-NLuc, respectively; top and bottom columns in (b), abar-NLuc/CLuc-WRKY40 and ABAR-NLuc/CLuc-WRKY40. The amounts of the expressed Luc proteins were assayed by immunoblotting with the goat anti-full-length firefly Luc antibody, which detect the N- and C-terminal firefly Luc fragments. The Luc amounts were used to assess the protein amounts of ABAR, abar, and WRKY40 in the tobacco leaves, and the data presented in (d) correspond to the assays in (A), and those presented in (e) correspond to (b). All the assays were repeated five times with similar results.