Design and Functional Characterization of a Novel Abscisic Acid Analog

The phytohormone abscisic acid (ABA) plays a crucial role in mediating plant growth and development by recruiting genetically redundant ABA receptors. To overcome its oxidation inactivation, we developed a novel ABA analog named 2′,3′-benzo-iso-ABA (iso-PhABA) and studied its function and structural characterization with A. thaliana ABA receptors. The (+)-iso-PhABA form showed much higher ABA-like activities than (+)-ABA including inhibitory effects on the seed germination of lettuce and A. thaliana, wheat embryo germination and rice seedling elongation. The PP2C (protein phosphatases 2C) activity assay showed that (+)-iso-PhABA acted as a potent and selective ABA receptor agonist, which is preferred to PYL10. In some cases, (−)-iso-PhABA showed moderate to high activity for the PYL protein inhibiting PP2C activity, suggesting different mechanisms of action of iso-PhABA and ABA. The complex crystal structure of iso-PhABA with PYL10 was determined and elucidated successfully, revealing that (+)-iso-PhABA was better coordinated in the same binding pocket compared to (+)-ABA. Moreover, the detailed interaction network of iso-PhABA/PYL10 was disclosed and involves hydrogen bonds and multiple hydrophobic interactions that provide a robust framework for the design of novel ABA receptor agonists/antagonists.


Materials and measurements
The 1 H and 13 C NMR spectra were recorded on a Bruker Avance DPX300 with tetramethylsilane as an internal standard. All NMR spectra were obtained using CDCl 3 as the solvent unless otherwise noted. GC-Mass was carried out on a 6890N GC-Agilent 5973N.
HPLC was carried US Agilent 1100. Mass spectra were obtained with a VG-ZAB-HS mass spectrometer. Mass spectra data are reported in mass to charge units (m/z). High-resolution mass spectra (HRMS) were recorded in ESI mode using Bruker Apex IV FTMS. Optical rotations were obtained from a Perkin Elmer 241MC polarimeter. Commercially available compounds were used in this work without further purification. The solvent tetrahydrofuran (THF) and benzene was dried by distillation from sodium and benzophenone. The To a stirred suspension of NaH (11.6g, 334mmol, 70% in oil) in THF (25mL) in a 500mL round bottomed flask, was added, 1-tetralone 5 (10.0 g, 69mmol) dissolved in dry THF (20mL). After stirring the mixture for 10 min at r.t., methyl iodide (11.1mL, 178mmol) was added slowly. The mixture was then heated to 40℃ for 30min, and stirring continued at r.t. 3h.

Bioassays.
The bioassays activities of the synthesized compounds were tested using our previously reported methods 1 .

Phosphatase activity assay
The phosphatase activity was measured by the serine-threonine phosphatase assay system (Promega V2460 kit). Each reaction was performed in a 45 L reaction buffer (20mMHepes pH 7.5, 150mM NaCl and 5mM MgCl 2 ) containing 3M HAB1, 5M PYLs protein and (+)/(-)-iso-PhABA of 10M concentration if required. After 30min at room temperature, 5L phosphorylated peptide substrate supplied with the Promega kit was added into the reaction system at 30°C for 25min. And then the reaction was terminated by addition of 50L molybdate dye/additive mixture, and the absorbance at 620nm was measured 30min later. The OD 620 value of the reaction without HAB1 was set as baseline while the phosphatase activity S7 of the reaction without PYLs was set as 100% for HAB1. Each reaction was repeated at least three times and the error bars indicated standard deviations.

MST
The microscalethermophoresis (MST) method has been described in detailelsewhere [2] . The

Crystallization and data collection
To get the PYL10-(+)-iso-PhABA complex crystals, (+)-iso-PhABA was mixed with purified PYL10 at 5:1 ratio and then incubated on ice overnight. The mixture was concentrated to about 10mg/mL.The crystallization screen conditions were from commercial kits (Hampton Research and Emerald Biosystems) and some self-made products. Initial trails were performed by sitting-drop vapor diffusion method at 20℃ and 4℃, respectively.
Crystallization-solution droplet was comprised of 1.0μL each reservoir solution and 1.0μL freshly purified target protein complex, which was equilibrated against 100μL each reservoir solution. The complex crystals appeared in a well solution contained 10% iso-propanol, 0.1M Na Hepes pH 7.5, 20% PEG4000. The crystal was transferred into well solution containing 20% glycol as cryo-protectant solution and flash-cooled in liquid nitrogen before collecting data.
To get the PYL10-(-)-iso-PhABA complex crystals, purified apo-PYL10 fragment was concentrated to about 10mg/mL for screening crystals. Apo-PYL10 native crystal appeared in the reservoir solution contained 25% PEG3350, 0.1MTris-HCl pH 8.5, 0.2M (NH 4 ) 2 SO 4 . The crystal was soaked into solution which was comprised of 1.0μL reservoir solution and 1.0μL 10mM (-)-iso-PhABA mother liquor. After seven days, the crystal was transferred into S8 solution containing 20% glycol as cryo-protectant solution and flash-cooled in liquid nitrogen before collecting data.
All the crystal data were collected at KEK beamline NE3A, SSRF beamline BL17U and BSRF beamline 1W2B. All the data were integrated and scaled with the HKL2000 suite of programs 3 . Data collection statistics are summarized in Table 1.