RESUMO
DELLA proteins are repressors of gibberellin signaling in plants. Our previous studies have indicated that gibberellin signaling is derepressed by SCF(GID2)-mediated proteolysis of the DELLA protein, SLENDER RICE1 (SLR1), in rice. In addition, the gibberellin-dependent increase of phosphorylated SLR1 in the loss-of-function gid2 mutant suggests that the SCF(GID2)-mediated degradation of SLR1 might be initiated by gibberellin-dependent phosphorylation. To confirm the role of phosphorylation of SLR1 in its gibberellin-dependent degradation, we revealed that SLR1 is phosphorylated on an N-terminal serine residue(s) within the DELLA/TVHYNP and polyS/T/V domain. However, gibberellin-induced phosphorylation in these regions was not observed in the gid2 mutant following the constitutive expression of SLR1 under the control of the rice actin1 promoter. Treatment with gibberellin induced both the phosphorylated and non-phosphorylated forms of SLR1 with similar induction kinetics in gid2 mutant cells. Both the phosphorylated and non-phosphorylated SLR1 proteins were degraded by gibberellin treatment with a similar half-life in the rice callus cells, and both proteins interacted with recombinant glutathione S-transferase (GST)-GID2. These results demonstrate that the phosphorylation of SLR1 is independent of its degradation and is dispensable for the interaction of SLR1 with the GID2/F-box protein.
Assuntos
Oryza/metabolismo , Proteínas de Plantas/metabolismo , Cromatografia por Troca Iônica , Giberelinas/metabolismo , Hidrólise , FosforilaçãoRESUMO
The phytohormone gibberellin (GA) controls growth and development in plants. Previously, we identified a rice F-box protein, gibberellin-insensitive dwarf2 (GID2), which is essential for GA-mediated DELLA protein degradation. In this study, we analyzed the biological and molecular biological properties of GID2. Expression of GID2 preferentially occurred in rice organs actively synthesizing GA. Domain analysis of GID2 revealed that the C-terminal regions were essential for the GID2 function, but not the N-terminal region. Yeast two-hybrid assay and immunoprecipitation experiments demonstrated that GID2 is a component of the SCF complex through an interaction with a rice ASK1 homolog, OsSkp15. Furthermore, an in vitro pull-down assay revealed that GID2 specifically interacted with the phosphorylated Slender Rice 1 (SLR1). Taken these results together, we conclude that the phosphorylated SLR1 is caught by the SCFGID2 complex through an interacting affinity between GID2 and phosphorylated SLR1, triggering the ubiquitin-mediated degradation of SLR1.
Assuntos
Proteínas F-Box/genética , Giberelinas/farmacologia , Oryza/genética , Proteínas de Plantas/genética , Complexos Ubiquitina-Proteína Ligase/genética , Proteínas Culina/genética , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutação , Oryza/efeitos dos fármacos , Oryza/metabolismo , Fosforilação , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Gibberellin (GA) regulates growth and development in plants. We isolated and characterized a rice GA-insensitive dwarf mutant, gid2. The GID2 gene encodes a putative F-box protein, which interacted with the rice Skp1 homolog in a yeast two-hybrid assay. In gid2, a repressor for GA signaling, SLR1, was highly accumulated in a phosphorylated form and GA increased its concentration, whereas SLR1 was rapidly degraded by GA through ubiquitination in the wild type. We conclude that GID2 is a positive regulator of GA signaling and that regulated degradation of SLR1 is initiated through GA-dependent phosphorylation and finalized by an SCF(GID2)-proteasome pathway.