1. Volume 10, Issue 9, Pages 1190-1205 (September 2017)
Modulation of ABA Signaling by Altering VxGΦL Motif of PP2Cs in Oryza sativa 
Seungsu Han, Myung Ki Min, Su-Youn Lee, Chae Woo Lim, Nikita Bhatnagar, Yeongmok Lee, Donghyuk Shin, Ka Young Chung, Sung Chul Lee, Beom-Gi Kim, Sangho Lee 
Molecular Plant 
Volume 10, Issue 9, Pages (September 2017)
DOI: /j.molp
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2. Figure 1 Sequence Analysis of PP2Cs to Reveal VxGΦL Motif.
(A) Nine ABA-signaling-related clade A OsPP2Cs contain a conserved motif, called “VxGΦL motif,” on the interaction interface with soluble ABA receptors. Red inverted triangle denotes the tryptophan residue (W259, “wedging tryptophan”) critical for the interaction with ABA receptors.
(B) Phylogenetic tree analysis shows that OsPP2C50 is the closest ortholog of AtHAB1, which has been extensively studied for its physiological role in stress-related ABA signaling pathway.
(C) The VxGΦL motif has a consensus sequence “VLGVL.” This figure was generated using WebLogo (Crooks et al., 2004).
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2
Determination of Key Residues in the VxGΦL Motif Involved in Interaction with OsPYL/RCARs.
(A and B) VxGΦL motif residues critical in the direct interaction of OsPP2C50 with OsPYL/RCAR3 and 10 were determined by the combination of alanine/hydrophile scanning using (A) GST pull-down assay or (B) phosphatase assay.
(C and D) The half-maximal inhibitory concentration (IC50) values for the phosphatase activities of the OsPP2C50 mutants used in the alanine/hydrophile scanning. The IC50 values are displayed in terms of the ABA concentration and reflect the ABA sensitivity of OsPYL/RCARs. n.d., not determined.
SI represents the OsPP2C50 wild-type and others the second and fourth residues of OsPP2C50 VxGΦL.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3 Crystal Structure of OsPP2C50:ABA:OsPYL/RCAR3.
(A) Overall structure of OsPP2C50:ABA:OsPYL/RCAR3. (Inset) Distances among residues around the VxGΦL motif. Red lines indicate intramolecular interactions of OsPYL/RCAR3 with S265 of OsPP2C50 and yellow lines with I267 of OsPP2C50.
(B–E) Residue-level details of the interface near the conserved VxGΦL motif of OsPP2C50 wild-type (B and D) and FM mutant (C and E) with OsPYL/RCAR3.
(F) The interactions of OsPP2C50 wild-type and K255A with OsPYL/RCAR3 investigated by phosphatase assay.
(G) Structural superposition of wild-type OsPP2C50 with FM mutant (green), complexed with OsPYL/RCAR3 (blue) and ABA. For clarity, only Cα atoms are shown. OsPYL/RCAR3 apparently undergoes a rigid-body rotation of 4.3° when complexed with OsPP2C50 FM mutant. The black and blue asterisks represent reference points for the rigid-body rotation of OsPYL/RCAR3.
Molecular Plant  , DOI: ( /j.molp )
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5. Figure 4
Conformational Dynamics of OsPP2C50:ABA:OsPYL/RCAR3 Complex and FM Mutant Revealed by HDX–MS.
(A) The HDX profile change upon complex formation is color coded onto the crystal structure of OsPP2C50:ABA:OsPYL/RCAR3 complex.
(B) The deuterium uptake levels from selected peptides from the complex are plotted.
(C) The differential HDX profile between wild-type (WT) and FM in their complex states is color coded onto the crystal structure of OsPP2C50:ABA:OsPYL/RCAR3 complex.
(D) The deuterium uptake levels from selected peptides from WT and FM complexes are plotted. Yellow sphere represents coordinated magnesium atoms, and stick represents ABA.
The data were derived from three independent experiments, and error bars represent the SEM. *p < 0.05.
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5
The Modulation of Protein–Protein Interactions between ABA Signaling Components via VxGΦL Motif of OsPP2C50.
(A) The comparison of activities between PP2C50 wild-type and FM mutant.
(B and C) The investigations of in vitro protein interactions by GST pull-down assay, which are of (B) OsPP2C50 wild-type (WT) and FM mutant with several OsPYL/RCAR proteins, and (C) OsPP2C6 and OsPP2C68's wild-type, SI, and FM mutant with OsPYL/RCAR3.
(D and E) Inhibition of phosphatase activity of OsPP2C50 wild-type and FM mutant by OsPYL/RCAR proteins at different ABA concentrations from 6 nM to 10 μM.
(F) Dissociation constants (KD) of the VxGΦL motif mutants of OsPP2C50 are shown with SEs, which were obtained from duplicates by BLI. Each mutant is represented such that only residues in the second and the fourth positions of the VxGΦL motif are noted in one-letter amino acid codes. For protein interaction studies with ABA effect, 0.1 mM ABA was added.
(G) Competitive interaction of OsPP2C50 with OsPYL/RCAR3 and OsSAPK10 was investigated by GST pull-down assay. “30min” indicates proteins were supplemented after 30 min pre-incubation of others at 4°C.
(H) The effect of VxGΦL motif alteration to SAPK10 was investigated by in vitro dephosphorylation assay with 2 μg of purified SAPK10.
Molecular Plant  , DOI: ( /j.molp )
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7. Figure 6
OsPP2C50 Mutants Modulate Differentially the Signaling Effects against OsPYL/RCAR3.
(A) All OsPP2C50 mutants critically suppress ABA signaling under ABA treatment conditions. OsPP2C50 mutants were introduced with ABA-responsive reporter and treated with ABA.
(B) Low amounts of OsPYL/RCAR3 cannot inhibit the suppression activity of OsPP2C50 mutants in ABA signaling. Both OsPYL/RCAR3 (4 μg of DNA) and OsPP2C50 mutants were introduced together with ABA-responsive reporter and treated with and without ABA.
(C) OsPP2C mutants show various suppression activities in ABA signaling depending on the amount of ABA receptor. OsPP2C50 mutants and variable concentrations of OsPYL/RCAR3 were introduced together and treated with 5 μM ABA. Error bars denote mean ± SD (n = 3). Values of y-axis indicate luminescence of firefly luciferase/Renilla luciferase × fLUC is driven by ABA-responsible promoter.
Molecular Plant  , DOI: ( /j.molp )
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8. Figure 7 ABA Sensitivity of OsPP2C50-OX Arabidopsis Transgenic Plants.
(A) RT–PCR analysis of OsPP2C50 gene expression. The expression levels of OsPP2C50 were analyzed in the leaves of 4-week-old transgenic plants. The Actin8 gene was used as an internal control, and the numbers in parentheses indicates the cycles of PCR.
(B) Germination rates of OsPP2C50-OX mutants and Col-0 plants on 0.5× MS medium supplemented with various concentrations of ABA. The numbers of seeds with emerged radicles were counted 2 and 4 days after planting.
(C and D) Seedling establishment of OsPP2C50-OX mutants and Col-0 plants exposed to ABA. The number of seedlings in each line with expanded cotyledons was counted (D) and representative photographs were taken at 9 days after planting (C).
(E and F) Root elongation of OsPP2C50-OX mutants and Col-0 in response to ABA. Root lengths of each plant were measured (F) and representative photographs were taken (E) at 5 days after sowing.
All data are the mean ± SE from three independent experiments. Different letters indicate significant differences in three independent experiments (ANOVA; p < 0.05).
Molecular Plant  , DOI: ( /j.molp )
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