Discovery of a new lead compound for plant growth retardants through compound library screening

Abstract

We screened a compound library to identify chemicals that retarded plant growth. From a chemical library of 9,600 compounds, one compound (BSA-1) inhibited the hypocotyls length of Arabidopsis seedlings grown in the dark with an IC₅₀ of approximately 0.35±0.05 µM. Investigation of the mechanisms underlying the action of BSA-1 suggested that this compound inhibits neither gibberellin biosynthesis nor brassinosteroid biosynthesis. Analysis of BSA-1 analogues provided clues about structurally important features for the development of new plant growth retardants.

Introduction

The unprecedented increase in food grain production after the 1960s is known as the Green Revolution. One of the remarkable technologies used in the Green Revolution was the introduction of dwarfing genes into wheat, thus increasing plant yields and providing a stronger and shorter stem that prevents the crop from falling over.^1,2) Currently, there is a tremendous need to control plant height in modern agriculture. For example, the management of high-quality turf on golf courses is challenging. To achieve the desired conditions (turfgrass at a height of 1 cm), daily mowing is required during the growing season, which costs a large sum of money annually worldwide.

Controlling plant size, which can be regulated genetically or chemically, is one of the most important aspects of modern crop production. Genetic control of plant height is primarily achieved by selecting shorter cultivars with dwarfing genes. There are many genes associated with semi-dwarf growth in wheat, some of which prevent the action of gibberellins (GA).³⁾ An alternate effective strategy for controlling plant height is the use of chemical plant growth retardants. These chemicals are applied to agronomic and horticultural crops to reduce unwanted longitudinal shoot growth without decreasing plant productivity. Indeed, altering the growth rate of turfgrass through the external application of chemicals has been the focus of intense research by turfgrass scientists for more than 50 years. In this context, great effort has been made to develop plant growth retardants.

Currently, there are numerous commercially available plant growth retardants. The biosynthesis of GA is a good target for plant growth retardants.⁴⁾ Paclobutrazol and uniconazole-P are used to control vegetative growth and enhance flowering and fruiting patterns to improve the economic yields of several crops. These plant growth retardants are potent inhibitors of ent-kaurene oxidase, thereby inhibiting the oxidation of ent-kaurene into ent-kaurenoic acid in the biosynthesis of GA.⁴⁾ Hence, searching for new plant growth retardants and exploring novel target sites for plant growth retardants are important for the agricultural sciences. Therefore, we carried out a systemic search for plant growth retardants. We discovered a new series of inhibitors that target brassinosteroid (BR) biosynthesis,^5–8) a plant growth-promoting hormone.⁹⁾ Available evidence indicates that BR biosynthesis inhibitors can significantly reduce the plant height of Arabidopsis, tobacco, maize and rice.^10–12) These observations indicate that BR biosynthesis inhibitors represent new plant growth retardants.

Discovering a new plant growth retardant lead compound is challenging. Recent advances in our knowledge of compound screening demonstrate that compound library screening is a useful technique for improving the chances of generating new lead compounds. Currently, a great number of small molecules have been produced, and various compound libraries are available. Here, we screened 9,600 small molecules to find new plant growth retardants based on the ability of the compounds to retard the stem elongation of Arabidopsis seedlings grown in the dark. We report the discovery of benzenesulfonamide derivatives as a new lead compound for plant growth retardants.

Materials and Methods

1. Chemical compound library and chemicals for biological studies

The chemical compound library was obtained from the Open Innovation Center for Drug Discovery (The University of Tokyo, Tokyo, Japan). A diverse subset of 9,600 compounds was assigned as a core chemical library with varieties of structural diversity. The structure of the compound was identified by liquid chromatography-mass spectrometry (LC-MS), and the purity was checked by the signal of the evaporative light-scattering detector (ELSD).¹³⁾ Analogues of BSA-1 of the highest grade available were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and they were of the highest grade available. Stock solutions of the test compounds were dissolved in DMSO at a concentration of 100 mM and stored at −30°C. The other reagents were of the highest grade, and they were purchased from Wako, Pure Chemical Industries, Ltd. (Tokyo, Japan).

2. Plant materials and growth conditions

Seeds of Arabidopsis (ecotype Columbia) were purchased from Lehle Seeds (Round Rock, TX, USA). Seeds used for the 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 1/2 Murashige and Skoog (MS) salt added to 96-well plates (Fukaekasei Co., Ltd., Kobe, Japan) with or without chemicals. Plants were grown under dark conditions in a growth chamber with or without chemicals. The biological activities of the test compounds were measured 5 days after the seeds were sowed. Stock solutions of all of the chemicals were dissolved in DMSO in a designed growth media at 0.1% (v/v), as previously described.⁶⁾

3. Screening of plant growth retardants

Screening of plant growth retardants was performed in a 96-well plate. A solution of 5 µL of the compound (10 µM; final concentration) and 150 µL of the plant growth media containing 1/2 MS salt and 1% solidified agar were added to each well. Arabidopsis seedlings were germinated and grown in the dark as described above. The biological activities of the test compounds were examined visually by measuring the length of the hypocotyls. Active compounds were subjected to second screening using a previously described method.⁶⁾

Results and Discussion

In the postembryonic development of higher plants, light signals activate photoreceptors to induce photomorphogenesis and de-etiolation and to inhibit hypocotyl elongation, the opening of the apical hook of cotyledons, the induction of greening, and the elongation of leaf primordia. In the absence of light, the elongation of the hypocotyl and the root is not inhibited, and the apical hook of cotyledons is maintained. The biological properties of Arabidopsis seedlings grown in the dark are useful for evaluating the activity of plant growth retardants.

To identify small molecules with plant growth retardant activities, we screened a diverse set of 9,600 synthesized chemicals established by the Open Innovation Center for Drug Discovery (The University of Tokyo, Tokyo, Japan). The seeds were placed in 96-well plates containing 1/2 MS agar medium and individual chemicals at 10 µM final concentrations. Plants were grown in the dark for 5 days at 22°C and examined visually for decreased hypocotyl lengths (Fig. 1).

Fig. 1. Screening for plant growth retardants. Seeds of Arabidopsis were grown for 5 days in the dark in 96-well plates in medium containing 1/2 MS and 9,600 individual compounds. Seedlings with short hypocotyls were selected and subjected to additional screening to establish repeatability. The chemical structure of BSA-1 is shown.

Thirty chemicals decreased the lengths of the hypocotyls of Arabidopsis seedlings grown in the dark. After retesting, one compound significantly inhibited stem elongation of Arabidopsis seedlings at a concentration of 10 µM (Compound 79617113, the structure is shown in Fig. 1). The IUPAC name for the compound is 2,5-dimethoxybenzenesulfonamide. We named the compound BSA-1. The hypocotyl length of plants grown in 1/2 MS medium was 15.6±0.2 mm, while that of BSA-1 treated plants was 2.8±0.1 mm (Fig. 1). To determine at which concentration BSA-1 most effectively retards stem elongation of Arabidopsis seedlings, we grew the plants in BSA-1 concentrations ranging from untreated controls to 100 µM in the dark (Fig. 2). The concentration of BSA-1 that caused 50% inhibition of hypocotyl length (IC₅₀) was 0.35±0.05 µM.

Fig. 2. Effect of BSA-1 on the inhibition of hypocotyl length of Arabidopsis seedlings grown in the dark. Arabidopsis seedlings were grown in 1/2 MS media containing BSA-1 in the dark for 5 days. The concentration of BSA-1 was 0.1, 1, 10, 100 µM. Average hypocotyl length at day 5 of treatment is shown (n>15). Error bars represent the standard deviation. Experiments were duplicated to establish repeatability.

To test whether BSA-1 inhibits BR biosynthesis or affects other plant growth-promoting hormones, such as GA, we co-treated plants with 10 µM of BSA-1 and 1 µM of GA or 10 nM of brassinolide (BL). Neither GA nor BL rescued BSA-1-induced dwarfism of Arabidopsis seedlings (Fig. 3). This result indicates that the ability of BSA-1 to inhibit stem elongation of Arabidopsis seedlings is not due to the inhibition of GA or BR biosynthesis.

Fig. 3. Responses of Arabidopsis seedlings to inhibitor treatments in the presence of BR and GA. Arabidopsis seedlings were grown in 1/2 MS media containing BSA-1 (1 µM) in the dark for 5 days. From left: without chemical treatment, co-application of BR (10 nM), and co-application of GA (1 µM). Average hypocotyl length at day 5 of treatment is shown (n>15). Error bars represent the standard deviation. Experiments were duplicated to establish repeatability.

To further determine the chemical structure–activity relationship of BSA-1 that is responsible for the retardation of Arabidopsis seedlings, five analogues of BSA-1 were developed and tested in a biological assay (the structures of BSA analogues are shown in Table 1). The IC₅₀ value of non-substituted benzenesulfonamide (BSA-2) was greater than 100 µM (Table 1), indicating that the chemical structure of benzenesulfonamide needs appropriate substituents on the benzene ring to exhibit potent inhibitory activity. Introducing a methyl group at position 4 of the benzenesulfonamide (BSA-3) did not promote the inhibitory activity either, displaying an IC₅₀ value greater than 100 µM (Table 1). Introduction of an amino group (BSA-4) or a chlorine atom (BSA-5) to the benzenesulfonamide skeleton at position 4 did not significantly promote the inhibitory activity of this synthetic series. However, introducing two electron-withdrawing fluorine atoms at positions 2 and 5 of benzenesulfonamide (BSA-6) significantly enhanced its inhibitory activity on stem elongation of Arabidopsis seedlings, with an IC₅₀ value of 15±3 μΜ. Although the inhibitory potencies of the analogues of BSA-1 used for the structure–activity relationship studies were not greater than that of BSA-1, further chemical structural optimization of this synthetic series may lead to the discovery of novel plant growth retardants.

Table 1. Inhibition of stem elongation of Arabidopsis seedlings by BSA analogues

R-	Compound ID.	IC50 (μM)
2,5-Dimethoxy	BSA-1	0.35±0.05
H	BSA-2	>100
4-Methyl	BSA-3	>100
4-Amino	BSA-4	95±6
4-Chloro	BSA-5	92±6
2,5-Difluro	BSA-6	15±3

Arabidopsis seedlings were grown on 1/2 MS media containing BSA analogues in the dark for 5 days. The concentration of BSA analogues was 0.1, 1, 10, 100 μM. Average hypocotyls length at day 5 of treatment is shown (n>15). Experiments were duplicated to establish repeatability.

Acknowledgment

This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This study was supported in part by the Akita Prefectural University President’s Research Project to K. Oh.

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