Dominant and pleiotropic effects of a GAI gene in wheat results from a lack of interaction between DELLA and GID1

Abstract

Dominance, semidominance, and recessiveness are important modes of Mendelian inheritance. The phytohormone gibberellin (GA) regulates many plant growth and developmental processes. The previously cloned semidominant GA-insensitive (GAI) genes Reduced height1 (Rht1) and Rht2 in wheat (Triticum aestivum) were the basis of the Green Revolution. However, no completely dominant GAI gene has been cloned. Here, we report the molecular characterization of Rht-B1c, a dominant GAI allele in wheat that confers more extreme characteristics than its incompletely dominant alleles. Rht-B1c is caused by a terminal repeat retrotransposons in miniature insertion in the DELLA domain. Yeast two-hybrid assays showed that Rht-B1c protein fails to interact with GA-INSENSITIVE DWARF1 (GID1), thereby blocking GA responses and resulting in extreme dwarfism and pleiotropic effects. By contrast, Rht-B1b protein only reduces interaction with GID1. Furthermore, we analyzed its functions using near-isogenic lines and examined its molecular mechanisms in transgenic rice. These results indicated that the affinity between GID1 and DELLA proteins is key to regulation of the stability of DELLA proteins, and differential interactions determine dominant and semidominant gene responses to GA.

Figure 1.

The phenotypes of NIL-Rht-B1c plants. A, Photographs of plants with NIL-Rht-B1a and NIL-Rht-B1c. Bar = 20 cm. B to F, Comparison of agronomic traits: plant height (B); seeds of plants with NIL-Rht-B1a and NIL-Rht-B1c (C); preharvest sprouting (D); tiller angle (E); and heading date (F). Student’s t test was used to generate P values. [See online article for color version of this figure.]

Figure 2.

Homology cloning of Rht-B1c. A, Amplification of the full lengths of Rht-B1a, Rht-B1b, and Rht-B1c. B, Comparison of gene structures of Rht-B1a and Rht-B1c. The thick black bars represent the coding sequence with the predicted translation start site (ATG) and stop code (TGA); the thin line represents the intron; and the dashed boxes represent the 90-bp insertion at the transcriptional level. gDNA, Genomic DNA.

Figure 3.

Architecture of DELLA domains of Rht-B1a and Rht-B1c. A, Comparison of amino acid sequences of DELLA domains among Rht-B1a, Rht-B1b, and Rht-B1c. The translated Rht-B1c protein contains an additional 30-amino acid insertion in the DELLAALGYKV motif. Asterisks represent the GID1 recognition sequence. B, A 3D model based on template 2ZSH (GA₃-GID1A-DELLA). C, A 3D model based on template 2ZSI (GA₄-GID1A-DELLA). In B and C, Rht-B1a is shown at left and Rht-B1c is shown at right. The DELLA motif of Rht-B1c formed three parts, residues 38 to 75, 76 to 89, and 90 to 141. αA, αΒ, αC, αD, and αE represent five α-helices. DELLAALGYKV (green) and VHYNP (yellow) motifs are essential for GID1 binding; the insertion α-helix (αF) of Rht-B1c is located within loop A-B, which is highlighted in red. [See online article for color version of this figure.]

Figure 4.

Structure of new TRIM. A, Schematic diagram of the structure of CAAS-TRIM-6B. PBS, Primer-binding site; PPT, polypurine tract; TDR, terminal direct repeat; TSD, target site duplication. B, Identification of TRIMs in different wheat cultivars using PCR. AB, T. durum (cv Langdon); Tz23, Taizhong 23; CS, Chinese Spring; H10, Hanxuan 10; L14, Lumai 14. C, Linkage map of CAAS-TRIM-6B and its surrounding markers on wheat chromosome 6B. [See online article for color version of this figure.]

Figure 5.

Gross morphology of transgenic rice plants. A transgenic Nipponbare plant (right) expressing Rht-B1b (middle) and Rht-B1c (left and top left corner) under the control its native promoter has semidwarf and extreme dwarf stature, respectively. Bar = 10 cm; bar in top left corner = 2.5 cm. [See online article for color version of this figure.]

Figure 6.

Loss of interaction activity with TaGID1 results in GA insensitivity. A, Morphologies of 10-d-old Rht-B1a, Rht-B1b, and Rht-B1c wheat seedlings treated with 50 μL L⁻¹ GA₃ (right) or without GA₃ (left). Bar = 5 cm. B, Comparison of seedling lengths of 10-d-old Rht-B1a, Rht-B1b, and Rht-B1c plants treated with (gray bars) or without (black bars) 50 μL L⁻¹ GA₃. Data were measured as means of 30 seedlings at each time with three replicates. Significance is as follows: * P ≤ 0.05, ** P ≤ 0.01. C, Relative variances of Rht-B1a, Rht-B1b, and Rht-B1c seedlings. The formula for variance is as follows: relative variance = (length of seedling with GA₃ − length of seedling without GA₃)/length of seedling without GA₃ × 100%. Significance is as follows: ** P ≤ 0.01. D and E, GA-mediated induction of α-amylase activity in Rht-B1a, Rht-B1b, and Rht-B1c seeds. A concentration of 1 μm GA₃ was applied to the plates. Sector I, Rht-B1a; sector ІІII, Rht-B1b; sector ІІІIII, Rht-B1c. F, Interaction between TaGID1 and Rht-B1. Y2H assays were performed using TaGID1 as bait and Rht-B1a, Rht-B1b, and Rht-B1c as prey. Left, growth of yeast strain AH109 transformants on −His−Ade plates; right, β-galactosidase activity detected in liquid assay with yeast strain AH109 transformants. To measure relative lacZ activity, three independent experiments were carried out. Five individual transformants were used each time. [See online article for color version of this figure.]