Abstract
Regulation of abscisic acid (ABA) signaling is crucial in balancing responses to abiotic stresses and retaining growth in planta. An ABA receptor (PYL/RCAR) and a protein phosphatase (PP2C), a co-receptor, form a complex upon binding to ABA. Previously we reported that the second and fourth positions in the VxGΦL motif of PP2Cs from Oryza sativa are critical in the interaction of PP2Cs with PYL/RCARs. Considering substantial effects of the VxGΦL motif on ABA signaling outputs, further comprehensive characterization of residues in the second and fourth positions are required. Here we surveyed the second and fourth positions of the VxGΦL motif by combination of biochemical, structural and physiological analyses. We found that the fourth position of the VxGΦL motif, highly conserved to small hydrophobic residues, was a key determinant of the OsPP2C50:OsPYL/RCAR interactions across subfamilies. Large hydrophobic or any hydrophilic residues in the fourth position abrogated ABA responsiveness. Analysis of crystal structures of OsPP2C50 mutants, S265L/I267V (“LV”), I267L (“SL”) and I267W (“SW”), in complex with ABA and OsPYL/RCAR3, along with energy calculation of the complexes, uncovered that a bulky hydrophobic residue in the fourth position of the VxGΦL motif pushed away side chains of nearby residues, conferring side-chain rotameric energy stress. Hydrophilic residues in this position imposed solvation energy stress to the PP2C:PYL/RCAR complex. Germination and gene expression analyses corroborated that OsPP2C50 AS and AK mutants modulated ABA responsiveness in Arabidopsis. Our results suggest that ABA responsiveness could be fine-tuned by the fourth position of the VxGΦL motif on PP2Cs.
Key message
We comprehensively surveyed the VxGΦL motif to find that the fourth position, highly conserved to small hydrophobic residues, was critical in regulating ABA responsiveness.
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Acknowledgements
We thank the staff members at beamline 5C, Pohang Accelerator Laboratory for technical assistance in crystallographic data collection. This work was supported by the Next-Generation BioGreen 21 program (PJ01367602) through the Rural Development Agency and the Basic Science Research Program (NRF-2017R1D1A1B03032185, NRF-2018R1A2B6004367 and NRF-2019R1A6A7076041) and the Science Research Center Program (SRC-2017R1A5A1014560) through the National Research Foundation of Korea (NRF) Grants. Accession numbers: Diffraction data and coordinates have been deposited to the protein data bank with accession numbers 5ZCG (LV mutant), 5ZCL (SL mutant) and 5ZCH (SW mutant). The accession number of the wild-type structure is 5GWP.
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SL conceived research plans and supervised experiments. SH designed, performed most of the experiments and analyzed the data. JL, YL and THK performed some experiments and analyzed the data. SH, THK and SL wrote the article with contributions from all the authors.
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Han, S., Lee, Jy., Lee, Y. et al. Comprehensive survey of the VxGΦL motif of PP2Cs from Oryza sativa reveals the critical role of the fourth position in regulation of ABA responsiveness. Plant Mol Biol 101, 455–469 (2019). https://doi.org/10.1007/s11103-019-00916-9
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DOI: https://doi.org/10.1007/s11103-019-00916-9