Article Figures & Data
Figures
- Figure 1.
Gross Morphology and GA Responsiveness of Sgd1.
(A) Gross morphology of Sgd-1 at the juvenile stage. gid1-8 (left), Sgd-1 (middle), and wild-type plants (T65; right) were grown for 4 weeks. Arrowheads represent the uppermost positions of the 3rd leaf sheath. Bar = 5 cm.
(B) Length of the 3rd leaf sheath of gid1-8 (left), Sgd-1 (middle), and T65 (right) grown for 4 weeks. Data are means ± sd, n = 14.
(C) Gross morphology of Sgd-1 at heading stage. gid1-8 (left), Sgd-1 (middle), and T65 (right) were grown for 3 months. Bar = 10 cm.
(D) Plant height of gid1-8, Sgd-1, and T65 grown for 3 months. Height was measured from the ground surface to the ear-neck node. Data are means ± sd, n = 14.
(E) Dose dependency of 2nd leaf sheath elongation of gid1-8, Sgd-1, and T65 grown on GA3 for 3 weeks. Data are means ± sd, n = 10.
- Figure 2.
Sgd-1 Is Caused by an Intragenic Mutation at the GID1 Locus.
(A) Schematic structure of GID1 indicating the positions of the gid1-8 mutation (L45→F), which is present in both gid1-8 and Sgd-1, and the Sgd-1 mutation (P99→S), which is present only in the Sgd-1 suppressor mutant. Amino acid residues shared with HSL, such as HGG and GXSXG, are shown within red boxes. The residues corresponding to the catalytic triad of HSL, S, D, and V, are indicated by filled circles.
(B) Linkage analysis between the Sgd-1 phenotype and the P99S mutation in the M2 generation of Sgd-1. The P99S mutation is caused by an SNP (C295T), which introduced a cleaved amplified polymorphic sequence polymorphism after digestion with HaeIII. Plants homozygous for the wild type or mutant allele at this SNP are designated as W or M, respectively, and heterozygous plants are designated as H. Plants showing the dwarf (D) phenotype of gid1-8 are designated as D, and plants showing the taller (T) phenotype of Sgd-1 are designated as T. All plants carrying the M and H genotypes showed the Sgd-1 phenotype (T), while plants carrying the W genotype showed the gid1-8 phenotype (D), indicating complete linkage between the Sgd-1 phenotype and the P99S mutation. M.M., molecular marker.
- Figure 3.
Effect of Substitutions of the 99th Pro in Rice GID1 on Its GA-Dependent or GA-Independent Interactions with SLR1.
(A) SLR-interacting activity of mutated GID1s, GID1WT, and the vector control (vec) in Y2H assays. Black bars indicate activity in the presence of 10−4 M GA4; gray bars indicate activity in the absence of GA4. Data are means ± sd, n = 3. Equal expression level of mutated GID1s and GID1WT in yeast cells was confirmed by immunoblot analysis (see Supplemental Figure 6A online).
(B) Y2H assays using GID1P99A or GID1WT as bait and SLR1 as prey in the presence of various concentrations of GA4. β-Gal activity was determined by a liquid assay with yeast strain Y187 transformants (means ± sd; n = 3).
(C) The structures of various truncated SLR1 proteins.
(D) Y2H assays using GID1P99A or GID1WT as bait and the mutated SLR1s as prey, with or without 10−4 M GA4. β-Gal activity detected in a liquid assay with yeast strain Y187 transformants (means ± sd; n = 3). Black bar indicates activity in the presence of 10−4 M GA4; gray bar indicates activity in the absence of GA4. Equal expression level of mutated SLR1s in yeast cells was confirmed by immunoblot analysis (see Supplemental Figure 6E online).
- Figure 4.
Physicochemical Analysis of the Interaction between GID1P99A and SLR1 (E4-R125) without GA.
(A) GID1WT–SLR1 (E4-R125) interaction without GA4. CM5 sensor chips upon which GST-SLR1 (E4-R125) was immobilized were injected with solutions containing 8, 9.5, 11, or 12.5 μg/mL Trx·His-GID1WT. RU, resonance units.
(B) GID1P99A–SLR1 (E4-R125) interaction without GA4. CM5 sensor chips upon which GST-SLR1 (E4-R125) was immobilized were injected with solutions containing 8, 9.5, 11, or 12.5 μg/mL Trx·His-GID1P99A.
(C) Various kinetic values; ka (association rate), kd (dissociation rate), and KD (kd/ka, dissociation constant), of GID1P99A–SLR1 (E4-R125) and GID1WT–SLR1 (E4-R125) interactions in the absence of GA4.
- Figure 5.
Physicochemical Analysis of the Interaction between GID1P99A and GA4 Using SPR.
(A) GID1WT–GA4 interaction. CM5 sensor chips upon which GST-GID1WT was immobilized were injected with solutions containing 0.0625, 0.125, 0.25, 0.5, and 1 μM GA4. RU, resonance units.
(B) GID1P99A–GA4 interaction. CM5 sensor chips upon which GST-GID1P99A was immobilized were injected with solutions containing 0.0625, 0.125, 0.25, 0.5, and 1 μM GA4.
(C) Various kinetic values; ka (association rate), kd (dissociation rate), and KD (kd/ka, dissociation constant), of GID1P99A–GA4 and GID1WT–GA4 interactions.
- Figure 6.
GA-Independent GA Signaling by GID1P99A in Planta.
(A) Top: Amount of GID1s produced in pAct-GID1WT (4 lines), pAct-GID1P99A (4 lines), and the vector control (Vec.) plants in gid1-4 background estimated by immunoblot analysis using an anti-GID1 antibody.
Bottom: Gross morphology of pAct-GID1WT and pAct-GID1P99A plants in gid1-4 background grown under normal conditions for 3 weeks. gid1-4 plants transformed with the vector control (Vec.) and wild-type (T65) plants are also shown. Bar = 10 cm.
(B) Same genotypes as in (A), grown with 10−6 M uniconazol for 3 weeks. Bar = 10 cm.
(C) Top: Amount of GID1s produced in pAct-GID1WT (three lines) and pAct-GID1P99A (three lines) plants in a gid1-3 cps1-1 background estimated by immunoblot analysis using an anti-GID1 antibody.
Bottom: Gross morphology of pAct-GID1WT and pAct-GID1P99A plants in a gid1-3 cps1-1 background grown under normal conditions for 3 weeks. Bar = 5 cm.
(D) Dose dependency of leaf sheath elongation of pAct-GID1WT and pAct-GID1P99A plants in a gid1-3 cps1-1 background grown in GA3 for 1 week (mean ± sd; n = 3).
- Figure 7.
Replacement of the At GID1b Loop with the At GID1a Loop Abolishes GA-Independent GID1–GAI Interaction.
(A) Y2H assay using mutated At GID1s as bait and GAI as prey in the presence or absence of GA4. In At GID1b (1a-loop), the entire loop region of GID1b (from S84 to T103) was replaced with the corresponding region from At GID1a. In At GID1bR90P, At GID1bR90P and H91H, and At GDI1bH91P, amino acids Arg-90 and/or His-91 were replaced with the corresponding residues from At GID1a, as indicated. β-Gal activity was determined by a liquid assay with yeast strain Y187 transformants (means ± sd; n = 3). Equal expression level of bait proteins in yeast cells was confirmed by immunoblot analysis (see Supplemental Figure 6B online).
(B) Various kinetic values; ka (association rate), kd (dissociation rate), and KD (kd/ka, dissociation constant), of At GID1a– and At GID1b–GA4 interactions by SPR.
- Figure 8.
Some Dicot GID1b-Type Proteins Show GA-Independent Interaction with Arabidopsis GAI.
(A) Phylogenetic analysis of dicot GID1s. Protein names of poplar GID1s defined by Mauriat and Moritz (2009) are shown next to each slash. A text file alignment used in this analysis is available as Supplemental Data Set 2 online.
(B) Y2H assay using various dicot GID1s and the mutated GID1s as bait and GAI as prey in the presence or absence of GA4. In soybean GID1b-2 (1a-1-loop) and soybean GID1b-2 (1a-2-loop), the entire loop region of soybean GID1b (from S82 to K102) was replaced with the corresponding region from soybean GID1a-1 and soybean GID1a-2, respectively. β-Gal activity was determined by a liquid assay of yeast strain Y187 transformants (means ± sd; n = 3). Equal expression level of bait proteins in yeast cells was confirmed by immunoblot analysis (see Supplemental Figure 6C online).
- Figure 9.
Molecular Model for Formation of the GA–GID1P99A–SLR1 Complex.
(A) In typical GID1s, such as OsGID1WTand At GID1a, GID1 cannot interact with DELLA protein in the absence of GA. When GA comes into its binding pocket, the lid closes on the binding pocket, which allows the interaction between GID1 and the DELLA/TVHYNP domains of DELLA protein. This interaction between GID1 and DELLA protein leads to degradation of DELLA protein (for instance, SLR1) and allows the derepressed state of GA responses.
(B) In certain GID1s, such as Os GID1P99S and P99A, At GID1b, and soybean GID1b-2 and -3, the lid tends to be closed even in the absence of GA, which promotes the interaction with DELLA protein. The closed state of the GID1P99A lid causes lower accessibility and slower dissociation of GA4. These properties of the receptor lead to GA hypersensitivity in the formation of the GID1–GA–DELLA protein complex. The interactions between GID1 and DELLA protein both with and without GA may allow the derepressed state of GA responses.
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