Binding of GID1 to DELLAs promotes dissociation of GAF1 from DELLA in GA dependent manner

Abstract

Gibberellins (GAs) are important phytohormones for plant growth and development. DELLAs are members of the plant-specific GRAS protein family and act as repressors of GA signaling. DELLAs are rapidly degraded in the presence of GAs. GA-GID1-DELLA complexes are recognized and ubiquitinated by the SCF(SLY) complex. The sleepy1 (sly1) F-box mutant exhibits dwarfism and low-germination phenotypes due to high accumulation of DELLAs. Overexpression of GID1 in the sly1 mutant partially rescues these phenotypes without degradation of DELLAs suggesting that proteolysis independent regulation of DELLAs exists in GA signaling. But the molecular mechanisms of non-proteolytic regulation of DELLA are largely unknown. Recently we identified a DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 also interacts with co-repressor TOPLESS RELATED (TPR) in nuclei. DELLAs and TPR act as coactivator and corepressor of GAF1, respectively. GAs converts the GAF1 complex from transcriptional activator to repressor via degradation of DELLAs. The overexpression of ΔPAM, lacking of DELLAs binding region of GAF1, partially rescue dwarf phenotypes of GA deficient or GA insensitive mutant. In this study, we investigate the relationship between non-proteolytic regulation of DELLAs and GA signaling via DELLA-GAF1 complex using modified yeast two-hybrid system.

Keywords: DELLA; GAF1; GID1; SLY1; TOPLESS; gibberellin; proteolysis; signaling; transcription factor.

Figure 1.

A co-activator model for GA signaling. Under GA deficient conditions, DELLAs are stable and localized in nuclei. DELLAs interact with GAF1, and exhibiting high transcriptional activity with GAF1. In the presence of GA, DELLAs are degraded via the 26S proteasome pathway. The GAF1-TPR complex exhibits transcriptional repression activity. GA-induced functional conversion of GAF1 complex in plants depends on the degradation of coactivator DELLAs. The ga1–3 or gai-1 mutant accumulate DELLA proteins at a higher level. ΔPAM cannot bind DELLA but can interact with TPR. ΔPAM protein acts as a constitutive repressor with TPR and promotes GA signaling.

Figure 2.

GID1 promote the dissociation of GAF1 from DELLA in GA-dependent manner. GAF1 fused with the GAL4 DNA-binding domain, GAI and RGA fused with the GAL4 activation domain and resulting β-galactosidase activity was detected in the presence or absence of GID1 and with or without 10⁻³ M GA₃. To avoid the suppression of transactivation activity of DELLAs by the masking of GA-GID1 to DELLA motif in yeast, we use GAL4 activation domain fused to DELLAs as prey protein. GAF1 interact with DELLAs, GAI and RGA, in yeast. The binding of GID1 promote the dissociation of GAF1 from DELLA in presence GA condition. Data are means±SD, n = 3 “vec” indicates empty vector using as negative control. Asterisks represent Student's t test significance compared with BD-GAF1 (*P < 0.05).

Figure 3.

Non-proteolytic regulation model for GA signaling. The sly-1 mutants accumulate DELLA proteins at a higher level. But the binding of GA-GID1 to DELLA promotes the dissociation of GAF1 from DELLA. The dissociated GAF1 from DELLAs could form a repressor complex with TPR and promote GA signaling.