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. 2019 Oct 22;116(43):21893-21899.
doi: 10.1073/pnas.1913532116. Epub 2019 Oct 9.

GIGANTEA Gates Gibberellin Signaling Through Stabilization of the DELLA Proteins in Arabidopsis

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Free PMC article

GIGANTEA Gates Gibberellin Signaling Through Stabilization of the DELLA Proteins in Arabidopsis

Maria A Nohales et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Circadian clock circuitry intersects with a plethora of signaling pathways to adequately time physiological processes to occur at the most appropriate time of the day and year. However, our mechanistic understanding of how the clockwork is wired to its output is limited. Here we uncover mechanistic connections between the core clock component GIGANTEA (GI) and hormone signaling through the modulation of key components of the transduction pathways. Specifically, we show how GI modulates gibberellin (GA) signaling through the stabilization of the DELLA proteins, which act as negative components in the signaling of this hormone. GI function within the GA pathway is required to precisely time the permissive gating of GA sensitivity, thereby determining the phase of GA-regulated physiological outputs.

Keywords: GIGANTEA; circadian clock; gating; gibberellin signaling; output oscillations.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
GI interacts with the DELLA proteins. (A and B) Yeast 2-hybrid (Y2H) assays showing interaction of GI and DELLA proteins. Bait and prey constructs were cotransformed into yeast cells. SD-WL, minimal medium lacking Trp and Leu; SD-WLH, selective medium lacking Trp, Leu, and His, which was supplemented with 50 mM 3AT. X-gal, qualitative β-galactosidase activity results obtained from the X-gal assay. (B) Quantitation of β-galactosidase activity (Miller units) for every pair of bait and prey proteins indicated (n = 4). Values represent means ± SEM [***P < 0.001; **P < 0.01; *P < 0.05; n.s., not significant Tukey’s multiple-comparison test relative to the pExpAD502 control vector]. (C) In vitro pull-down assays showing the interaction between GI and DELLAs (RGA, GAI, and RGL3). Proteins were expressed in an in vitro transcription and translation system. (D) In vivo coimmunoprecipitations in Arabidopsis transgenic seedlings expressing HA-GI (from the 35S promoter) and GFP–RGA (from an endogenous promoter fragment) tagged protein versions.
Fig. 2.
Fig. 2.
RGA is stabilized by GI, and GI function is required to shape oscillations in RGA protein accumulation. (A) Representative Western blot showing the accumulation of RGA-GFP in N. benthamiana leaves treated with 25 µM MG-132 or in the presence or absence of GI-HA. Protein levels were normalized against HA-GFP levels. (B) Quantitation of 3 biological replicates of the experiment shown in A (mean ± SEM; *P < 0.05; n.s., not significant Tukey’s multiple comparison test). Protein levels were normalized against HA-GFP levels. (C) Accumulation of GFP–RGA across SD photo-cycles in WT (Col-0), gi-2, and GIox backgrounds. ACTIN levels were used for normalization, and the quantitation of 3 biological replicates is shown (mean ± SEM; ***P < 0.001, **P < 0.01 Bonferroni post hoc test following 2-way ANOVA). White and gray shadings represent day and night, respectively. (D) Representative confocal images of 10-d-old SD-grown seedlings expressing GFP–RGA in WT (Col-0), gi-2, and GIox backgrounds taken from the upper part of the hypocotyl at ZT12. (E) Hypocotyl length measurements from WT (Col-0), GIox, rga-29;gai-td1, and GIox;rga-29;gai-td1 seedlings grown for 7 d in SDs (mean ± SEM, n = 24 to 36; ***P < 0.001; **P < 0.01; n.s., not significant Tukey’s multiple comparison test).
Fig. 3.
Fig. 3.
GI stabilizes RGA in the context of its GA-GID1–mediated degradation. (A) Interaction between Flag-GID1A and HA-RGA in the presence of increasing quantities of cMyc-GI (0.25×, 0.5×, 1×, 2×, and 4×). Proteins were expressed in a TnT in vitro expression system and immunoprecipitated with anti-Flag antibody. (B) Quantitation of the relative amount of HA—RGA coimmunoprecipitated with GID1A in every fraction from the experiment shown in A. (C) Degradation time course of GFP–RGA in WT (Col-0) and gi-2 mutants. The 10-d-old SD-grown seedlings were treated at ZT7 with 100 µM GA3 and 200 µg/mL cyclohexamide. ACTIN levels were used for normalization. (D) Quantitation of the relative amount of GFP–RGA in every fraction from the experiment shown in C. Protein levels were normalized against ACTIN levels. (E) Quantitation of RGAΔ17-GFP accumulation in N. benthamiana leaves treated with 25 µM MG-132 or in the presence of GI–HA. Protein levels were normalized against HA–GFP levels. Values represent mean ± SEM (n = 3) (n.s., not significant Tukey’s multiple comparison test). (F) Hypocotyl length measurements from WT (Ler), gi-3, gai-1, and gi-3;gai-1 seedlings grown for 7 d in SDs (in gray, mean ± SEM, n = 16 to 20; ***P < 0.001 Tukey’s multiple comparison test).
Fig. 4.
Fig. 4.
GI is required to adequately gate GA signaling at night. (A and B) GA3 and PAC dose–response curves for WT (Col-0) and gi-2 mutant seedlings. Plants were grown for 7 d under SD conditions with increasing concentrations of GA3 (0, 0.1, 1, and 10 µM) (A) or for 3 d in the dark in the presence of increasing concentrations of PAC (0, 0.02, 0.2, and 2 µM) (B). Values represent means ± SEM (n = 24 to 36) (***P < 0.001; n.s., not significant Bonferroni post hoc test following 2-way ANOVA). (C) Hypocotyl length (measured as the difference between GA-treated and mock-treated seedlings) of seedlings grown for 6 d under SD conditions in the presence of 0.2 µM PAC and treated with 1 µM GA4 at different ZTs (mean ± SEM, n = 25) (n.s., not significant; **P < 0.01; ***P < 0.001 Bonferroni post hoc test following 2-way ANOVA). (D) Hypocotyl length of HS::gai-1D lines in WT (Col-0), gi-2, and GIox backgrounds treated with heat at ZT12 to induce the expression of the GAI dominant negative version gai-1D (brown bars) compared to nontreated controls (gray bars). Values represent mean ± SEM (n = 33 to 39) (*P < 0.05; ***P < 0.001 Tukey’s multiple comparison test). HS, heat-shock–treated plants.
Fig. 5.
Fig. 5.
Model of GI action in the gating of GA signaling. As GI accumulates during the day, it stabilizes the DELLAs by hindering access of the GA receptor GID1A, the expression of which is circadian-regulated and high in the evening. Progressive degradation of GI during the evening enables the degradation of the DELLA proteins and the expression of GA-responsive genes, including growth-promoting genes.

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