Fig. 5.

ANT potently blocks ABA signaling in planta. (A) ANT increases transpiration in multiple species. Infrared images of 6-wk-old tomato seedlings (UC82), 3-wk-old wheat seedlings (Patwin 515), and 3-wk-old wild-type Arabidopsis (Col-0) plants treated with ANT (100 µM) and imaged by thermography either 2 h (tomato and wheat) or 48 h (Arabidopsis) postapplication. Statistical analyses were performed using unpaired t tests (n = 8 for tomato, n = 9 for wheat, and n = 10 for Arabidopsis). Error bars indicate SD. *** indicates P < 0.0001. (B) ANT treatments phenocopy abi1-1 mutant phenotypes. Infrared images of wild-type Arabidopsis plants continuously exposed to ANT (100 µM) or mock compared to mock-treated abi1-1. Images were collected 3 wk after continuous exposure. Dunnett tests were used to obtain multiplicity-adjusted P values for treatment effects relative to mock treatments (n = 12). (C and D) ns indicates not statistically significant. ANT blocks osmotic stress–induced gene expression. (C) Five-day-old marker line seedlings were treated with 400 mM mannitol for 6 h, with coapplication of either 2.5 µM ANT, PanME, AA1, or mock treatment. (Scale bars, 0.5 mm.) The full dataset for this experiment is presented in SI Appendix, Fig. S11. (D) Comparison of transcript levels of RD29B and MAPKKK18 (normalized to PEX4) measured by qRT-PCR of 8-d-old Arabidopsis seedlings pretreated with either dimethyl sulfoxide (DMSO) or 25 µM ANT for 4 h and then exposed to either DMSO (−) or 25 µM ANT(+) in the presence (+) or absence (−) of 20% PEG for another 6 h. * indicates P < 0.05 for indicated comparisons. (E) ANT accelerates seed germination. Seed germination was monitored for seeds plated on 1/2 Murashige & Skoog (MS), 0.7% agar plates containing DMSO (mock treated) or 100 µM ANT (barley) or 25 µM ANT (tomato). Time-response data were fit to a log-logistic model using the drc package to infer ET₅₀ values; however, barley under mock treatment never reached 50% germination. ET₅₀ significantly differs between ANT and mock tomato treatments (two-sample t test, P < 0.01, n = 3) and the percent germination after 4 d significantly differs between ANT and mock-treated Palmella landrace (two-sample t test, P = 0.001, n = 3 for ANT, n = 6 for mock). Data for both landraces tested (Morex and Palmella) are shown in SI Appendix, Fig. S13. (F) ANT alleviates the effects of thermoinhibition in Arabidopsis. The ET₅₀ values inferred from this experiment are shown in SI Appendix, Fig. S13 and were generated by quantifying germination over time for seeds plated on 1/2 MS, 0.7% agar plates containing DMSO (mock treated) or 30 µM test chemicals either at 22 °C or after exposure to heat stress (37 °C for 48 h). Error bars indicate the SEM. Under heat stress, ANT and fluridone ET₅₀ values differ from mock (pairwise two-sample t test with Bonferroni correction, P < 0.001, n = 5 for ANT and fluridone, n = 4 for mock), and under control conditions ANT ET₅₀ significantly differs from fluridone and mock treatments (pairwise two-sample t test with Bonferroni correction, P < 0.001, n = 5 for ANT and fluridone, n = 4 for mock).