Fig 2: Hierarchical organization of the DWA1–RGL2 signaling network.(A) Genetic hierarchy of germination. Seeds germinated on ½ MS ± 1 µM PAC or ABA, scale bar = 0.2 mm. (B) Quantification of hormone sensitivity. Germination rates after 2 days are analyzed. Data are means ± SD, n = 3 independent biological repeats; different letters indicate P < 0.05 by two-way ANOVA followed by Tukey’s test. Source data are available online for this figure.

Fig 3: PYL receptors directly stabilize RGL2 through hormone-modulated interactions.(A) Bimolecular fluorescence complementation (BiFC) visualization of PYL–RGL2 interactions. YFP signals from Nicotiana benthamiana co-expressing YNE-RGL2 and PYLs-YCE were visualized, scale bar = 50 µm. (B) In vitro pull-down of PYL–RGL2 complexes. GST-PYLs were incubated with MBP-RGL2, captured with glutathione resin, and immunoblotted. (C) Hormonal regulation of PYL–RGL2 interactions. Luciferase complementation imaging (LCI) quantification in N. benthamiana expressing nLUC-PYLs and cLUC-RGL2 treated with 10 µM ABA or GA₃. Data are means ± SD (n = 3 independent biological replicates). Significant differences were determined by two-way ANOVA. (D) PYL-mediated suppression of GA-induced RGL2 degradation. Five-day-old seedlings (genotypes indicated) expressing MYC-RGL2 were pretreated with 10 µM paclobutrazol (PAC) for 60 min, then challenged with 10 µM GA. Right: densitometric quantification (Data represent means from three independent biological repeats, two-way ANOVA followed by Tukey’s test). Source data are available online for this figure.

Fig 4: Receptors gate the DWA1–RGL2 interaction.(A) PYL4 restrains DWA1-catalyzed ubiquitination. N. benthamiana leaves co-expressing RGL2-GFP, Flag-UBQ10, and MYC-DWA1 were infiltrated ± PYL4-mCherry and 50 µM MG132 was added 12 h before harvest. (B) Cell-free degradation of recombinant MBP-RGL2. Purified MBP-RGL2 was incubated with extracts from 5-day-old WT, 35S:Flag-DWA1, 35S:PYL4-GFP or 35S:Flag-DWA1 35S:PYL4-GFP seedlings in the presence of 10 µM GA₃ for the indicated times. Rubisco (Ponceau S) is a loading control. (C) Co-IP confirmation of PYL4 interference. MYC-DWA1 and RGL2-GFP were co-expressed ± PYL4-mCherry; anti-MYC IP, immunoblot as indicated. (D) PYL4 disrupts the DWA1–RGL2 association (LCI). N. benthamiana expressing nLUC-RGL2 + cLUC-DWA1 + mCherry/PYL4-mCherry. Data are means ± SD, n = 3 independent biological replicates. Significant differences were determined by one-way ANOVA followed by Tukey’s test. (E) Same interaction monitored by BiFC. YNE-RGL2 + DWA1-YCE + mCherry/PYL4-mCherry; scale bar = 25 µm. (F) GA-dependent enhancement of DWA1–RGL2 interaction. nLUC-RGL2 + cLUC-DWA1 expressed for 2 days with 50 µM MG132, then treated ± 10 µM GA₃ for 3 h. Data are means ± SD, n = 3 independent biological repeats. Significant differences were determined by unpaired t-test (P = 0.00012). (G) GID1 requirement for GA-induced complex assembly. Co-IP in wild-type and gid1b/c protoplasts expressing MYC-DWA1 and RGL2-GFP ± 10 µM GA₃. gid1b/c: GID1-deficient mutant. (H) GID1 promotes DWA1-mediated RGL2 ubiquitination in vivo. MYC-DWA1, RGL2-GFP and Flag-UBQ10 expressed ± GID1a/b-mCherry. Tissues were treated with 50 µM MG132 and 10 µM GA₃ (12 h), followed by anti-GFP immunoprecipitation and anti-Flag immunoblotting. (I) GID1 dependence of GA-accelerated RGL2 turnover. MBP-RGL2 protein was incubated with extracts from the 5-day-old WT, 35S:Flag-DWA1, gid1b/c or 35S:Flag-DWA1 gid1b/c seedlings plus 10 µM GA3 for the indicated times. Source data are available online for this figure.

Fig 5: Microscale thermophoresis (MST) analysis of RGL2–receptor interactions.Purified, fluorescently labeled sfGFP-RGL2 (0.5 µM) was titrated with the indicated GST-fused receptors in the presence of 100 µM GA (for GID1a/b) or ABA (for PYR1, PYL4, PYL11). Curves show fraction bound versus receptor concentration; data are means ± SD from three technical replicates. Apparent Kd values: GID1a: 3.86 ± 1.47 µM, GID1b: 4.64 ± 0.69 µM, PYR1: 43.95 ± 25.82 µM, PYL4: 22.74 ± 12.60 µM, PYL11: 17.01 ± 8.60 µM (± Kd Confidence). GID1 receptors exhibit ~4-fold higher affinity for RGL2 than PYLs under the same conditions.