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We carried out the biochemical evaluation of the target site of propiconazole in BR biosynthesis. Applying BR biosynthesis intermediates to Arabidopsis seedlings grown in the presence of propiconazole under dark condition, we found that the target site of propiconazole in BR biosynthesis can be identified among the C22 and C23 side chain hydroxylation steps from campestanol to teasterone. Using differential spectra techniques to determine the binding affinity of propiconazole to CYP90D1, which is responsible for C23 hydroxylation of BR, we found that propiconazole induced typical type II binding spectra in response to purified recombinant CYP90D1 and the Kd value was found approximately 0.76 μM.
Plant architecture is an important factor affecting grain yields of crops. An unprecedented increase in food grain production in the last century, which has been called as “Green Revolution," was achieved by growing lodging-resistant, semi-dwarf varieties of wheat and rice.1,2 Controlling of plant architecture is one of the most important aspects in modern agriculture. Selecting shorter cultivars with dwarfing genes have made great contributions.3 There are many genes associated with semi-dwarf wheat, some of which prevent the action of gibberellins (GA).4 Additionally, the application of chemicals that targeting GA biosynthesis represents a feasible way to control the unwanted longitudinal shoot growth of crops.5 Paclobutrazol (Pac) 6 and uniconazole (Ucz)7 are widely used plant growth regulators (their structures are shown in Fig. 1). Both compounds inhibit GA biosynthesis thereby reducing plant height and inducing the morphological characteristics of GA deficient mutants.6-8
Recent progress on the studies of brassinosteroid (BR) functions has indicated that BRs is a good target for bioengineering of high yield of crops.9 Analysis the BR-deficient mutants gave evidences that BRs play a key role in promoting plant growth.10 Also, BRs are important signals in response to environmental cues.11,12 BRs are essential hormones that involve in cell elongation and sex determination.10,11,13 Deficient mutants BR biosynthesis display growth defects.14-16 Hence, specific inhibitors of BR biosynthesis could be used as a plant growth regulator for enhancing crop production.
The biosynthetic pathway of BRs was established using tracer experiments with labeled biosynthesis precursors of brassinolide (BL) in periwinkle (Catharanthus roseus) cell lines17 together with the analysis of the endogenous levels of BRs in BR-deficient mutants .18 Use of genetic and GC-MS techniques, the metabolic pathway of BRs biosynthesis has been established (Fig. 2).19 The C-22 hydroxylation of campesterol (CR) was catalyzed by DWF4/CYP90B1.18 The C-3 dehydrogenation of steroid skeletons is believed to be catalyzed by CPD/CYP90A1.20 CYP90C1/ROT3 and CYP90D1, were shown as C-23 hydroxylases.21 Arabidopsis CYP85A1 and CYP85A2 were found to catalyze the C-6 oxidation reaction.22
The first synthetic triazole-type of BR biosynthesis inhibitors, brassinazole (Brz, chemical structure shown in Fig. 1), was discovered by Asami and co-workers.23,24 The target site(s) of Brz have been identified as DWARF4 which catalyzed the C22 hydroxylation in BR biosynthesis.24 Application use of Brz demonstrated that specific inhibitors of BR biosynthesis are useful to reveal the functions of BR in various plant species. 25,26 Also, they can be used for the isolation and characterization of BR signaling genes.27,28 To develop new inhibitors targeting novel target in BR biosynthesis, we recently reported a new series of BR biosynthesis inhibitors. Using ketoconazole as a molecular scaffold,29 the structure-activity relationship studies revealed yucaizol (the structure is shown in Fig. 1) as currently the most potent inhibitor of BR biosynthesis found to date.30-32 The use of YCZ-18, an analog of yucaizol (the structure is shown in Fig. 1), demonstrated that yucaizol is a specific inhibitor of BR biosynthesis that binds to purified recombinant CYP90D1.33 In the course of these works, we established several assay systems that can be used to determine the target site of small molecules in BR biosynthesis. One experiment system which is based on using purified recombinant protein of CYP90D1 allows us to conduct in vitro evaluation the inhibitory activity of small molecules against BR biosynthesis. Another assay system, which is based on the use of BR biosynthesis intermediators, can be used to determine the inhibitory effects of small molecules in BR biosynthesis in vivo.33 With these experimental system, we resently demonstrated that fenarimol, a pyrimiding type funjicide, inhibits BR biosynthesis.34 Currently, numerous triazole compounds have been shown to inhibit P450s. Recently, Hartwig and his coworkers reported that propiconazole (Pcz), 1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1,2,4-triazole, (the structure is shown in Fig. 1), exhibits potent inhibitory activity on BR biosynthesis in Arabidopsis and maize through inducing phenocopy of BR deficient mutants.35 The target site of Pcz in BR biosynthesis is lacked of elucidation.
To explore the target site of Pcz in BR biosynthesis, we conducted a biochemical assessment of the target site of Pcz in BR biosynthesis through determination of the binding affinity of Pcz to the BR biosynthesis enzyme CYP90D1 together with the analysis of the effects of BR biosynthesis intermediates on Pcz-treated Arabidopsis grown in the dark.
Propiconazole, GA3 and campesterol were purchased from Wako Pure Chemical Industries, Ltd (Tokyo, Japan). Brassinolide, castasterone, teasterone were purchased from Burashino K.K. (Toyama, Japan), Campestanol was prepared by reduction of campesterol based on a method as described previously.36 All of the chemicals for biological studies, unless otherwise described, were dissolved in DMSO and stored at −30°C before use.
Seeds of Arabidopsis (Columbia ecotype) were purchased from Lehle Seeds (Round Rock, TX, USA). The seeds used for assay were sterilized in 1% NaOCl for 20 min and washed with sterile distilled water. Seeds were sown on a 1% solidified agar medium containing half Murashige and Skoog salt in agripots (Kirin Brewery. Co., Tokyo, Japan) with or without chemicals. Plants were grown under 16-h light (240 μmol. M-2 s-1) and 8-h dark conditions in a growth chamber. For the dark condition, agripots were wrapped in 4 layers of aluminum foil. The biological activities of the test compounds were measured 5 d after sowing the seeds. Stock solutions of all of the chemicals were dissolved in DMSO at a designed concentration and applied to growth media at 0.1% (v/v).
Arabidopsis full-length cDNA was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.37 The expression vector, pCold-GST, was obtained from Dr. C. Kojima of Osaka University.38 The DNA fragment encoding CYP90D1 mature protein was generated by PCR with forward primer 5′-AATCGAGCTCATGGACACTTCTTCTTCACTTTTG-3′ and reverse primer 5′-TTGACTGCAGTTATATTCTTTTGATCCAAATGGGT-3′. The PCR product was digested with SacI-PstI and was inserted into the pCold-GST expression vector. All of the constructed plasmids were transferred to the BL21 star (DE3) strain of E. coli (Invitrogen). The transformed cells were incubated in 10 ml of Luria broth containing 100 μl/ml of chloramphenicol overnight at 37°C. Next, 10 ml of pre-culture was incubated in 1000 ml of Luria broth containing 100 μl/ml of ampicillin at 37°C.
The expression and purification of recombinant CYP90D1 were performed as described previously.33 Protein measurements were performed using a Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and bovine serum albumin as a standard. The relative purity of recombinant CYP90D1 was estimated by SDS-polyacrylamide gel electrophoresis (12% polyacrylamide) and gel staining was performed using Coomassie brilliant blue R250.
Binding of Pcz to CYP90D1 was measured by optical difference spectroscopy of purified recombinant CYP90D1 using a Shimadzu UV3100 spectrophotometer as we previously described.33 The data obtained were used to calculate binding constants based on linear regression analysis. Spectral determinations were performed at least twice for each experiment, confirming the reproducibility with respect to the spectral profile and position of λmax and λmin.
To explore the target site of Pcz in BR biosynthesis, we first determined the effect of BR biosynthesis intermediates on the rescue of the Pcz induced dwarf phenocopy of dark grown Arabidopsis seedlings. Arabidopsis seedlings were treated with Pcz at a concentration of 5 μM together with co-application of commercially available BR biosynthesis intermediates. As shown in Fig. 3, the hypocotyl length of untreated controls was found to be approximately 12.2 ± 0.2 mm (Fig. 3G; 3H: ultraviolet bar). When the Arabidopsis seedlings treated with 5 μM Pcz, We found that the hypocotyl length of Arabidopsis seedlings was approximately 2.3 ± 0.1 mm (Fig. 3A; 3H empty bar). Following co-application of TE (10 μM, Fig. 3D, 3H: green bar), CS (100 nM, Fig. 3E, 3H yellow bar) and the final product BL (10 nM, Fig. 3F, 3H: black bar) in the growth media, the hypocotyl length of Arabidopsis seedlings was restored from 2.3 ± 0.1 to 9.6 ± 0.2, 6.4 ± 0.1 and 9.5 ± 0.2 mm, respectively. This result clearly indicates that the BR biosynthesis intermediates of TE, CS and BL reversed the dwarfism of dark grown Arabidopsis seedlings with the Pcz-treatment, implying Pcz does not inhibit the enzymes downstream of TE in BR biosynthesis (Fig. 2). However, co-application of CR (100 μM, Fig. 3B, 3H: red bar) or CN (100 μM, Fig. 3C, 3H: blue bar) did not restore the dwarfism of Arabidopsis. The hypocotyl length changed from 2.3 ± 0.1 to 3.2 ± 0.1 mm and from 2.3 ± 0.1 to 2.5 ± 0.1 mm, respectively. This result indicated that Pcz blocked the steps downstream of CN in BR biosynthesis. Taking these results together, Pcz inhibits the steps of BR biosynthesis from CN to TE, which is performed by CYP90B1, CYP90C1 and CYP90D1 (Fig. 2)21,25 Use of similar methods, Noguchi et al. determined the mechanism of BR deficient mutant of det2.39 In addition, this method was also used for the determination of the target site of Brz.40 Nevertheless, to characterize the target site of Pcz in BR biosynthesis inhibition, careful experiments should be carried out by determine the binding affinity of Pcz to purified BR biosynthesis enzymes.
Based on the results obtained from feeding experiments, we next assessed the target site responsible for Pcz in BR biosynthesis inhibition. We first cloned the genes of CYP90B1, CYP90C1 and CYP90D1 into the pCold-GST expression vector. Despite substantial efforts to optimize the expression conditions of these genes, CYP90B1 and CYP90C1 proteins were poorly expressed in soluble fractions in E. Coli expression system. Thus, we could not obtain the purified recombinant. However, In a condition as described in the experimental section, we successfully expressed CYP90D1 in soluble fraction and obtained the purified CYP90D1. Next, we determined the binding affinity of Pcz to CYP90D1.
Using optical difference spectra techniques, we determined the binding affinity of Pcz to CYP90D1. The addition of Pcz to the purified CYP90D1 protein induced a type II absorbance shift of the heme Soret band (Fig. 4A). The dissociation constant Kd was determined by titrating the observed spectral absorbance difference (ΔA437-A414) versus the concentration of Pcz (Fig. 4B). The double reciprocal plot for calculating Kd revealed that the dissociation constant for Pcz was found in the average of 2 experiments at 0.76 μM (Fig. 4B; Table 1).
In the present work, we used biochemical means to investigate the target site of Pcz in BR biosynthesis. The feeding of BR biosynthesis intermediates to Arabidopsis seedlings with Pcz-induced dwarfism provided evidence that Pcz targets the side chain hydroxylation located between campestanol and teasterone (Fig. 3). Analysis of the binding affinity of Pcz to C23 hydroxylase of CYP90D1 provided conclusive evidence that Pcz targeted the step(s) of the side chain hydroxylation of BR biosynthesis (Fig. 4). Because the soluble recombinant protein of CYP90B1 and CYP90C1 was poorly expressed in E. coli, we could not perform the experiment to determine the binding affinity of Pcz to CYP90B1 and CYP90C1. We expect application use of other expression system such as yeast expression system may yield soluble recombinant proteins of these genes. Nevertheless, regarding the structural similarity of Pcz and Brz-220 (see Fig. 1), a specific inhibitor of BR biosynthesis binds to CYP90B1,41 it is reasonable to conjecture that Prz binds to CYP90B1 together with the C23 hydroxylation enzyme (s) in BR biosynthesis.
Data obtained from the present work provided evidence for the first time on the biochemical elucidation of the target site of Pcz in BR biosynthesis. Our results clearly indicated that Pcz binds to CYP90D1. The feeding of BR biosynthesis intermediates provided evidence that Pcz targets side chain hydroxylation in BR biosynthesis.
No potential conflicts of interest were disclosed.
This study was supported in part by funding from the Akita Prefectural University President’s Research Project to K. Oh.