6B). loss. Three-week-old Col-0 plants were sprayed with the indicated chemicals. The loss of fresh weight was measured after 12 h of chemical treatment (= 3; error bars = se). F, Expression of ABA-responsive genes after chemical treatment (DMSO as mock). Col-0 seedlings were sprayed with the indicated chemicals for 6 h, after which gene expression levels were determined by quantitative real-time reverse Amyloid b-Protein (1-15) transcription-PCR (= 3; error bars = CAPN2 se). G, AA1 attenuates ABA-induced RD29a expression. Five-day-old pRD29-LUC transgenic seedlings were sprayed with the indicated chemicals, and the photographs were taken after 6-h chemical treatments. The concentrations of chemicals are shown as ABA/AA1 (m/m). ABA controls stomatal aperture and transpiration rate, which is one of Amyloid b-Protein (1-15) its crucial physiological roles that can be measured indirectly through the leaf heat (Kim et al., 2010; Gonzalez-Guzman et al., 2012). The leaf surface temperature increases when stomata close because of reduced evaporative cooling. The ABA-treated leaves displayed elevated leaf temperatures; this did not occur when the leaves were treated with AA1 at the same time (Fig. 1D). More importantly, AA1-treated leaves showed a decreased leaf temperature in an AA1 dose-dependent manner, indicating that AA1 can function in guard cells and can antagonize endogenous ABA (Fig. 1D). Consistent with the fact that AA1 increased transpiration (Fig. 1D), the AA1-treated leaves lost water more quickly (Fig. 1E). High salt concentration induces ABA biosynthesis and, hence, inhibits seed germination (Gonzalez-Guzman et al., 2012). To examine the effect of AA1 on high-salt responses, Arabidopsis seeds were sown on MS medium made up of 200 mm NaCl, or AA1 plus NaCl. As shown in Supplemental Physique S1, the AA1-treated seeds effectively resisted high salt. Collectively, AA1, as an effective ABA antagonist, functions in ABA-mediated seed germination and growth regulation. Whats more, AA1 could be a useful tool for the study of ABA signaling in both monocots and Amyloid b-Protein (1-15) dicots. AA1 Inhibits ABA-Responsive Gene Expression ABA signal transduction results in the up-regulation of stress-response genes (Cao et al., 2013). To characterize whether AA1 may affect ABA-induced gene expression, we detected the expression levels of and in seedlings under different chemical treatments. These two genes were dramatically activated by ABA treatment, but their expression was reduced when the seedlings were cotreated with AA1 and ABA (Fig. 1F). ABA-induced stress-response gene expression also can be monitored in transgenic plants made up of a firefly luciferase (promoter, made up of the DRE/C-repeat and ABRE elements (Ishitani et al., 1997; Cao et al., 2014). The resulting transgenic plants emit bioluminescence in response to cold, osmotic stress, or exogenous application of ABA (Ishitani et al., 1997). Amyloid b-Protein (1-15) ABA treatment enhanced the expression of = 3; error bars = se). IC50, Fifty percent inhibitory concentration. C, AA1 attenuates the ABA-dependent inhibition of HAB1 activity via various ABA receptors. Various PYR/PYL-HAB1 combinations were incubated with the indicated chemicals (2 Amyloid b-Protein (1-15) m ABA, 100 m AA1, or DMSO; = 3; error bars = se). D, Dose-dependent effects of AA1 on PYR/PYL-dependent inhibition of PP2C activity. HAB1 activity was tested with dimeric receptors (PYR1 and PYL2) or monomeric receptors (PYL5 and PYL10) with 2 m ABA and the indicated concentrations of AA1 (= 3; error bars = se). Next, the antagonist activity of AA1 was characterized by examining the way in which it reverses ABA-induced PP2C inhibition through PYR/PYL. All 13 Arabidopsis ABA receptors are activated after ABA binding, and ABA-bound receptors are responsible for PP2C inhibition. Hence, receptor activation can be monitored through PP2C activity inhibition. In the phosphatase assays, AA1 failed to activate any of the 13 receptors. This indicates that, unlike pyrabactin, which selectively activates (e.g. PYR1 and PYL1) or represses (e.g. PYL2) certain receptors, AA1 has no natural agonist activity (Fig. 2C). AA1 could repress all 13 receptors activated by ABA, revealed by its ability to reverse ABA-mediated PP2C inhibition, to certain levels (Fig. 2C). In addition, AA1 displayed unbiased antagonist activity to both dimeric receptors (PYR1 and PYL1CPYL3) and monomeric receptors (PYL4CPYL12; Fig. 2,.
