Abscinazole-E3M, a practical inhibitor of abscisic acid 8'-hydroxylase for improving drought tolerance

Abstract

Abscisic acid (ABA) is an essential phytohormone that regulates plant water use and drought tolerance. However, agricultural applications of ABA have been limited because of its rapid inactivation in plants, which involves hydroxylation of ABA by ABA 8'-hydroxylase (CYP707A). We previously developed a selective inhibitor of CYP707A, (-)-Abz-E2B, by structurally modifying S-uniconazole, which functions as an inhibitor of CYP707A and as a gibberellin biosynthetic enzyme. However, its synthetic yield is too low for practical applications. Therefore, we designed novel CYP707A inhibitors, Abz-T compounds, that have simpler structures in which the 1,2,3-triazolyl ring of (-)-Abz-E2B has been replaced with a triple bond. They were successfully synthesised in shorter steps, resulting in greater yields than that of (-)-Abz-E2B. In the enzymatic assays, one of the Abz-T compounds, (-)-Abz-E3M, acted as a strong and selective inhibitor of CYP707A, similar to (-)-Abz-E2B. Analysis of the biological effects in Arabidopsis revealed that (-)-Abz-E3M enhanced ABA's effects more than (-)-Abz-E2B in seed germination and in the expression of ABA-responsive genes. Treatment with (-)-Abz-E3M induced stomatal closure and improved drought tolerance in Arabidopsis. Furthermore, (-)-Abz-E3M also increased the ABA response in rice and maize. Thus, (-)-Abz-E3M is a more practical and effective inhibitor of CYP707A than (-)-Abz-E2B.

Figure 1. Structures of UNI, Abz-E2B and novel CYP707A inhibitors.

Figure 2. Synthesis of compounds 3–6.

Reagents and conditions: (i) 4- or 3-iodobenzaldehyde, K₂CO₃, Ac₂O, 100 °C; (ii) UV 365 nm, MeOH, room temperature (RT); (iii) NaBH₄, MeOH, RT; (iv) Pd(PPh₃)₂Cl₂, Cul, Et₃N, THF, RT; (v) NaH, 1-butanol, RT; (vi) NaH, 2-methoxyethanol, 60–80 °C.

Figure 3. Effects of (−)-Abz-T compounds on Arabidopsis seed germination compared with that of (−)-Abz-E2B.

Seed germination rates in the presence of 0.1 μM ABA and the indicated concentrations of azole inhibitors at a time when the germination rate of plants treated only with ABA was 80% (n = 3; error bars represent s.d.). Compound 6 is also referred to as Abz-E3M.

Figure 4. Effects of (−)-Abz-T compounds on rice seedling growth compared with the effects of UNI and (−)-Abz-E2B.

Seedlings grown on test media containing indicated concentrations of azole inhibitors for 7 d. Scale bars represent 10 mm. Compound 6 is also referred to as Abz-E3M.

Figure 5. Effects of (−)-Abz-E3M on the expression levels of Arabidopsis ABA-responsive genes.

Expression of MAPKKK18, RD29A and RD29B after treatment with (−)-Abz-E2B or (−)-Abz-E3M in the absence or presence of 0.25 μM ABA (n = 4, error bar represents s.d.). Ten-day-old seedlings of wild-type (Col accession) were incubated in solutions containing the chemicals for 6 h.

Figure 6. Effects of (−)-Abz-E3M on Arabidopsis drought tolerance.

(a,b) Three-week-old plants were treated with 50 μM of (−)-Abz-E3M and incubated overnight at 22 °C with 60% relative humidity. Thermal images of plants were obtained using a thermal imaging camera (TH9100WR, Nippon Avionics Co., Ltd.), and the average temperatures on leaf surfaces were determined with NS9200 software (n = 5, error bars represent s.d.). Representative images are shown in (a). (c) Stomatal apertures after the application of 50 μM (−)-Abz-E3M. After an overnight incubation, plants were placed at 22 °C with ~90% relative humidity for 1 h. Stomatal images were obtained using Suzuki’s universal micro-printing method. Values are means ± SEM (n = 5, ~30 stomata per individual leaf). (d) Effects of (−)-Abz-E3M on endogenous ABA levels. Four-week old plants grown in soil pots were treated with 50 μM (−)-Abz-E3M, and leaves were harvested after an overnight incubation. Values are means with s.d. (n = 6). (e) Four-week-old plants were subjected to drought stress by withholding water. Initially, 50 mL of 50 μM (−)-Abz-E3M containing 0.05 % Tween-20 (or a no chemical control treatment) was sprayed three times every other day on 135 Arabidopsis plants. Plants were rehydrated after a 12-d drought treatment, and their subsequent recovery statuses were photographed after 3 d. The number of surviving plants per total number of tested plants is inset in the photograph.

Figure 7. Effects of (−)-Abz-E3M on maize.

(a) Thermal images of the leaf surface temperatures of (−)-Abz-E3M-treated maize plants. (b) Leaf surface temperatures of (−)-Abz-E3M-treated maize plants. The stomatal conductance (c) and transpiration rate (d) in (−)-Abz-E3M-treated maize at different light intensities. (a–d) One-month-old plants were used in this experiment, and 10 mL of 100 μM (−)-Abz-E3M was sprayed per plant, with data obtained at 30,000 ± 1,500 lux, 41.5 ± 1.5 °C and 35 ± 5% relative humidity after an overnight incubation. Values are means with s.d. (n = 4 for leaf surface temperature, n = 3 for stomatal conductance and transpiration rate). (e) Effects of (−)-Abz-E3M on endogenous ABA levels. (f) Expression levels of ZmLEA, Zm.12309.1.S and ZmCOR410 after the (−)-Abz-E3M treatment. (e,f) Root parts of 2-week-old whole seedlings were dipped in 100 μM (−)-Abz-E3M chemical solution containing 0.05% Tween-20 overnight, and aerial shoot tissues were used for qRT-PCR and ABA measurement analyses. Values are means with s.d. (n = 3 for qRT-PCR, n = 5 for ABA measurement).