The Gibberellin Signaling Pathway Is Regulated by the Appearance and Disappearance of SLENDER RICE1 in Nuclei

Effect of GA₃ on the Subcellular Localization of SLR1-GFP.

(A) and (D) Confocal microscopic images of GFP fluorescence in young leaf sections from SLR1-GFP overexpressor lines under the control of the rice Actin1 promoter.

(B) and (E) Nuclei in the same cells as in (A) and (D) were stained with DAPI.

(C) and (F) Merged images of GFP and DAPI fluorescence.

Plants were grown with (+GA₃, [D] to [F]) or without (non-treat, [A] to [C]) 100 μM GA₃ for several days before GFP fluorescence analysis. Bars = 10 μm.

Effect of GA₃ on the Amount of SLR1.

(A) Complementation of the slr1 phenotype with SLR1 prom::SLR1-GFP. Introduction of SLR1 prom::SLR1-GFP rescued the slender phenotype (middle plant). slr1-1 (left) and wild-type (right) plants are shown as control plants.

(B) to (D) Confocal microscopic images of GFP fluorescence in young leaf sections from SLR1 prom::SLR1-GFP transgenic lines. To block GA biosynthesis, the transgenic rice seedlings were pretreated with 1 μM uniconazol ([C] and [D], +uni) and then treated with 100 μM GA₃ for 6 hr ([D], +uni, +GA₃ 6 hr). non-treat indicates normal growth conditions without any treatment (B). Bars = 10 μm.

(E) Protein gel blot analysis of the SLR1 protein. Rice seedlings were grown for 1 week under normal conditions (lane 2) or with 1 μM uniconazol (lane 1; uni). For the GA treatment, the seedlings treated with uniconazol then were sprayed with 100 μM GA₃ and collected after 6 hr (lane 3). The arrowhead and square indicate the protein bands corresponding to endogenous SLR1 and SLR1 nonrelated protein, respectively. Each lane contains 10 μg of total protein. As a loading control, the Coomassie brilliant blue (CBB) staining profile is shown.

Diagram of the Deleted Constructs for Domain Analysis of SLR1.

Each domain—DELLA, TVHYNP, Ser/Thr/Val-rich domain (polyS/T/V), LZ, nuclear localization signal (NLS), VHIID, PFYRE motif, and SAW motif—is indicated by different shading. The deleted SLR1 mutants were fused with the GFP coding sequence to generate overproducers for phenotypic analysis (see Figure 5) and subcellular localization studies (see Figure 6). The deletion points in each mutated SLR1 are shown below each box.

Gross Morphologies of 10-Day-Old Wild-Type and Transgenic Seedlings Overproducing the Truncated SLR1-GFP Proteins with GA₃ Treatment ([I] to [P]) or Nontreatment ([A] to [H]).

Plants were grown for 6 days under normal conditions and then treated with or without 100 μM GA₃ for another 4 days. Because ΔDELLA, Δspace, ΔTVHYNP, and ΔpolyS/T/V transgenic plants showed a severe dwarf phenotype and never produced any fertile flowers, we used T1 generation plants for the analyses. Asterisks in (I), (J), (N), (O), and (P) show the top of the elongated fourth leaf sheath. Bars = 1 cm.

Nuclear GFP Fluorescence Pattern in Young Leaves of Transgenic Plants.

The same plants shown in Figure 5 were used for analysis of the nuclear localization of SLR1-GFP. To confirm the nuclear localization of the GFP fluorescence, the positions of nuclei were always tested by DAPI staining. non-treat, nontreatment. Bars = 10 μm.

Effect of PolyS/T/V Deletion on the Regulation of GA Action.

(A) Protein gel blot analysis of the fusion proteins in an SLR1-GFP–overproducing plant (lane 1) and two independent lines of ΔpolyS/T/V-GFP–overproducing plants (lanes 2 and 3). Crude extracts were extracted from the shoot apices of each plant. Twenty micrograms of total protein was subjected to SDS-PAGE, electroblotted, and probed with an anti-HA antibody. The arrowhead and asterisk indicate the positions of intact SLR1-GFP and ΔpolyS/T/V-GFP proteins, respectively.

(B) Endogenous SLR1 protein level in a wild-type plant (lane 1), a SLR1-GFP plant (lane 2), and the ΔpolyS/T/V-GFP line 2 plant used in (A) (lane 3). Protein extracts were subjected to SDS-PAGE and probed with anti-SLR1 antibody. Each lane contains 20 μg of total protein. The circle shows the degraded protein derived from the ΔpolyS/T/V-GFP protein.

(C) Level of OsGA20ox transcript. RNA gel blot analysis was performed with total RNA (10 μg) isolated from the shoot apices of the same plants used in (B).

All of these samples were prepared from adult plants grown for 30 days.

Dimer Formation of SLR1 through the LZ Domain in the Yeast Two-Hybrid Assay.

For bait constructs (Bait), full-length SLR1, ΔLZ, or ΔC-Ter (see Figure 4) was fused with the G4BD. For the prey construct (Prey), the full-length SLR1 was fused with the G4AD. The relative lacZ activity of various combinations is presented. p53 and T-antigen (simian virus 40 T-antigen) were used as positive controls to evaluate relative binding affinity. For each pairwise combination, five individual transformants were used to measure relative lacZ activity. Error bars represent standard deviations.