The DELLA motif is essential for gibberellin-induced degradation of RGA

Abstract

RGA and GAI are homologous genes that encode putative transcriptional regulators that repress gibberellin (GA) signaling in Arabidopsis. Previously we showed that the green fluorescent protein (GFP)-RGA fusion protein is localized to the nucleus in transgenic Arabidopsis, and expression of this fusion protein rescues the rga null mutation. The GA signal seems to derepress the GA response pathway by degrading the repressor protein RGA. The GA-insensitive, semidominant, semidwarf gai-1 mutant encodes a mutant protein with a 17-amino acid deletion within the DELLA domain of GAI. It was hypothesized that this mutation turns the gai protein into a constitutive repressor of GA signaling. Because the sequences missing in gai-1 are identical between GAI and RGA, we tested whether an identical mutation (rga-Delta 17) in the RGA gene would confer a phenotype similar to gai-1. We demonstrated that expression of rga-Delta 17 or GFP-(rga-Delta 17) under the control of the RGA promoter caused a GA-unresponsive severe dwarf phenotype in transgenic Arabidopsis. Analysis of the mRNA levels of a GA biosynthetic gene, GA4, showed that the feedback control of GA biosynthesis in these transgenic plants was less responsive to GA than that in wild type. Immunoblot and confocal microscopy analyses indicated that rga-Delta17 and GFP-(rga-Delta 17) proteins were resistant to degradation after GA application. Our results illustrate that the DELLA domain in RGA plays a regulatory role in GA-induced degradation of RGA. Deletion of this region stabilizes the rga-Delta 17 mutant protein, and regardless of the endogenous GA status rga-Delta 17 becomes a constitutively active repressor of GA signaling.

Figure 1

Effect of GA₃ treatment on the phenotypes of control and transgenic lines. All lines are 36 days old and have been treated (+) or not treated (−) with GA₃ as indicated. All the lines except the hemizygous rga-Δ17 line are homozygous for the mutation and/or the transgene as labeled. homo, plants are homozygous for the transgene; hemi, plants are hemizygous for the transgene.

Figure 2

Final heights of plants in response to repeated applications of GA₃. All the lines except the hemizygous rga-Δ17 line are homozygous for the mutation and/or the transgene as labeled. Final heights of untreated (−) and GA-treated (+) plants are shown in black and gray, respectively. hemi, plants are hemizygous for the transgene. Means ± SE were measured for 8–12 plants per line.

Figure 3

Hypocotyl growth response to GA₃. All the lines except the hemizygous rga-Δ17 line are homozygous for the mutation and/or the transgene as labeled. Hypocotyl lengths of ga1-3 and rga-24/ga1-3 were compared with rga-Δ17 and GFP-(rga-Δ17) lines (A) and RGA and GFP-RGA overexpression lines (B). The curves are shown for GA₃ concentrations that give a linear response. The values plotted are the means ± SE of 12 seedlings measured. Some error bars are too small to be seen.

Figure 4

The rga-Δ17 protein is resistant to GA treatment. The blots contain total plant proteins (25 μg in A and 50 μg in B) extracted from 8-day-old seedlings after treatment with water (−) or GA (+) as labeled. (A) Affinity-purified rabbit anti-RGA polyclonal antibodies and a peroxidase-conjugated goat anti-rabbit IgG were used to detect the RGA (64-kDa) and rga-Δ17 (62-kDa) proteins. Control lane, 0.5 ng of nickel column-purified 65-kDa His-tagged RGA protein. The upper arrow with a question mark indicates the unknown protein that is present only in plants expressing the rga-Δ17 protein. (B) Rat anti-GFP polyclonal antibodies and a peroxidase-conjugated goat anti-rat IgG were used to detect GFP-RGA and GFP-(rga-Δ17) fusion proteins (91- and 89-kDa, respectively). The extra upper band in A and the additional lower band in B are nonspecific background proteins.

Figure 5

Effect of GA on the fluorescence in the roots of transgenic plants expressing the GFP-RGA and GFP-(rga-Δ17) fusion proteins. Transgenic seedlings were incubated for 2 h with water (+ H₂O) or 100 μM GA₃ (+ GA₃), and then fluorescence in root tips was visualized by confocal laser microscopy under an identical setting for all images.

Figure 6

Levels of GA4 mRNA in Ler, ga1-3, gai-1, rga-Δ17, and GFP-(rga-Δ17) line A. Shown is an autoradiogram of RNA blots containing 9 μg of total RNA isolated from 13-day-old seedlings with (+) or without (−) GA₃ treatment as labeled. The blot was hybridized with a labeled GA4 antisense RNA probe and then reprobed with a labeled 18S rDNA probe. The values under the blots indicate the relative amounts of GA4 mRNA after standardization using 18S rRNA as a loading control. The value of Ler (−GA₃) was arbitrarily set to 1.0.