Abscinazole-E2B, a practical and selective inhibitor of ABA 8'-hydroxylase CYP707A.

We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 8'-hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated logP value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 µM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (±)-Abz-E2B was a considerably weaker inhibitor than (±)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (±)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 µM, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 µM or higher concentration is required, although (±)-Abz-E2B may be useful as an alternative option at a lower concentration.

Dehydration tolerance in apple seedlings is affected by an inhibitor of ABA 8'-hydroxylase CYP707A.

The effects of an abscisic acid (ABA) 8'-hydroxylase inhibitor (Abz-F1) on ABA catabolism, stomatal aperture, and water potential were examined in apple seedlings under dehydration and rehydration conditions. In this study, 9-cis-epoxycarotenoid dioxigenase (MdNCED) and ABA 8'-hydroxylase (MdCYP707A) genes were isolated and their expressions were investigated under dehydration and rehydration conditions. The stomatal aperture decreased up to 4 h after spraying with Abz-F1 and the stomatal aperture in the Abz-F1-treated leaves was generally lower than that in the untreated control-leaves during the dehydration condition. Although the water potential in untreated control-leaves decreased with the progress of dehydration, it was maintained at a higher level in the Abz-F1 treated-leaves than in the untreated control-leaves during dehydration. Endogenous ABA concentrations increased with dehydration in both the Abz-F1 treated- and untreated-control-leaves, but the ABA levels in the Abz-F1 treated-leaves were higher than those in the untreated control-leaves throughout dehydration. In contrast, the phaseic acid (PA) concentrations in the Abz-F1 treated-leaves were lower than those in the untreated control-leaves during dehydration. The expressions of MdNCEDs in the Abz-F1 treated-leaves were lower than those in the untreated control-leaves regardless of the higher endogenous ABA concentrations. Moreover, the expressions of MdCYP707As in the Abz-F1 treated-leaves were also lower than those in the untreated control-leaves. Higher 50% effective concentrations (EC(50)) and ascorbic acid concentrations were observed in the Abz-F1 treated-leaves, which show that the oxidative damage under dehydration may be reduced by Abz-F1 application. These results suggest that prompt stomata closure is required for survival under dehydration, and Abz-F1 application may therefore be of practical use. The increase of endogenous ABA, which induced prompt stomata closure in Abz-F1 treated-leaves may depend on inhibition of the expression of MdCYP707As. Furthermore, the results showed the close relationship between MdNCEDs and MdCYP707As on ABA catabolism.