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Discovery of Shoot Branching Regulator Targeting Strigolactone Receptor DWARF14

DWARF14 (D14) is a strigolactone receptor that plays a central role in suppression of shoot branching, and hence is a potential target to increase crop productions and biomass. Recently, we reported a fluorescence turn-on probe, Yoshimulactone Green (YLG), which generates a strong fluorescence upon the hydrolysis by D14-type strigolactone receptors. Herein, we applied a YLG-based in vitro assay to a high-throughput chemical screening and identified a novel small molecule DL1 as a potent inhibitor of D14. DL1 competes with endogenous strigolactones, thereby increasing the number of shoot branching in a model plant Arabidopsis as well as in rice. Thus, DL1 is expected to be useful not only as a tool to understand the biological roles of D14 receptors in plant growth and development, but also as a potent agrochemical to improve the crop yield.


Plant materials
The Arabidopsis thaliana Columbia (Col) accession was used as wild type. The nipponbare (lowland-adapted japonica) was used in rice branching assay. 35S::AtD14-GFP plasmid was constructed through LR recombination (Thermo Fisher Scientific) with pGWB5 and used for transformation into atd14-1 by floral dip method (ref. 1).

Plant culture and treatment
For plant assays using Arabidopsis, the seeds were sterilized, stored in the dark at 4 °C for a few days, transferred to 0.5

Preparation of recombinant proteins
The procedure for the purification of His-tagged proteins was described previously (ref. 2). Aliquots of purified recombinant AtD14 and AtHTL were kept at -80 °C until use.

YLG-based assays
In vitro YLG hydrolysis assays were conducted using 1 µg of recombinant AtD14 in 100 µL of the reaction buffer (100 mM HEPES, 150 mM NaCl, pH 7.0) with 0.2% or 1.1% DMSO on a 96-well black plate (Greiner). After YLG was incubated with recombinant AtD14 for 1 hour, the fluorescence intensity was measured by spectraMax i3 (Molecular Devices) at excitation by 480 nm and detection S13 by 520 nm. For high-throughput screening, 10 µM of library compounds (1% DMSO) were co-incubated with 1 µM of YLG. The high-throughput experiments were supported by using automated liquid handling system (Sciclone ALH3000, Perkin Elmer). 800 library compounds were selected from the ITbM chemical library (20101 compounds, 1 mM DMSO solution, specially prepared reagent for biochemical research, Nakalai-tesque, Code: 13445-45) purchased from ChemDiv, Inc (San Diego, CA, USA) and Enamine, Inc (Kiev, Ukraine). For dose-dependent inhibition assays, DL1 and other hit compounds at the range between 0.1 to 10 µM were treated with 1 µM YLG in the presence of recombinant AtD14. IC 50 values were calculated by a curve-fitting program in Kaleidagraph software. For Lineweaver-Burk plot analysis, AtD14-mediated hydrolysis of YLG at the range between 0.1 to 40 µM was conducted in the presence or absence of DL1. K m and V max values were calculated by using the linear least square method.

Intrinsic tryptophan fluorescence assay
For investigating the affinity of small molecule to AtD14, 10 µM of recombinant AtD14 was incubated with small molecules at the range of 0 to 20 µM in 100 µL of the reaction buffer (10 mM HEPES, 150 mM NaCl, pH 7.0) with 0.1% DMSO on a 96-well black plate (Greiner). For investigating the affinity of small molecule to AtHTL, 10 µM of recombinant AtHTL was incubated with small molecules at the range of 0 to 250 µM in 100 µL of the reaction buffer (20 mM HEPES, 150 mM NaCl, 5% glycerol, pH 7.0) with 1% DMSO on a 96-well black plate (Greiner). After incubation at room temperature for 30 min, intrinsic fluorescence was measured at excitation by 288 nm and detection by 320-440 nm. The fluorescence intensity at 340 nm was plotted against the concentration of small molecules, and the dissociation constant (K d ) was calculated by a curve-fitting program in Kaleidagraph software.

Seed germination assay
Freshly harvested seeds of Arabidopsis were suspended in distilled water and aliquoted in 48-well plates. racemic-GR24 and DL1 were added to the seeds (DMSO 0.2%). The seeds were incubated under continuous light at 26 °C. After the incubation for 36 hours, radicle emergences were counted under a dissecting microscope.