벼의 개화 신호 전달 경로에서 Response Regulator 단백질들의 기능 연구

Abstract

Flowering time is elaborately controlled by various environment factors, but is ultimately induced by florigens such as FT or FT-like molecules. In rice (Oryza sativa), Ehd1 is a major inducer of florigen gene expression. However, the molecular mechanism by which this protein regulates downstream genes is poorly understood. For studying its molecular function, Ehd1 knockout mutants were generated by the CRIPSR/Cas9 method. In the T0 generation, approximately 91% of the tested plants showed delayed flowering under short- or long-day conditions. In contrast, transgenic rice plants over-expressing Ehd1 flowered extremely early. In those transgenics, transcript levels for Hd3a and RFT1 were dramatically increased. The tissue-specific expression pattern of Ehd1 was elucidated by creating transgenic plants that harbored a chimaeric molecule between the Ehd1 promoter and the GUS reporter. Similar to the patterns for Hd3a and RFT1, GUS was preferentially expressed in the phloem. Although Ehd1 was ectopically over-expressed in pHd3a::GUS plants, the GUS signal was still restricted to the phloem tissue. Taken together, these results suggest that Ehd1 directly regulates Hd3a and RFT1 in those phloem tissues. Although Ehd1 is highly homologous to members of the type-B response regulator family in the cytokinin signaling pathway, its precise molecular mechanism is not well-understood. Here, the C-terminal portion of the protein containing the GARP DNA-binding (G; maize GOLDEN2, the ARR B-class proteins from Arabidopsis, and Chlamydomonas Psr1) domain promoted flowering when over-expressed. Moreover, the N-terminal portion of Ehd1, carrying the receiver (R) domain, caused delayed flowering due to the inhibition of endogenous Ehd1 activity. In addition, the C-terminal stretch showed transactivation and the G domain had a nuclear localization signal. The Ehd1 protein formed a homomer via a 16-amino-acid region in the interdomain between R and G. Based on site-directed mutagenesis analyses, it was apparent that phosphorylation of the Asp-63 residue within the R domain induces the homomerization of Ehd1, which is crucial for Ehd1 activity. Overexpression of Ehd1 D63E, which mimics the constitutive phosphorylation state, also leads to extremely early flowering. Exogenous treatment with cytokinin also delayed flowering. A cytokinin-inducible type-A response regulator (RR) -- OsRR1 -- physically interacted with Ehd1 to form a heterodimer. In addition, OsRR1-overexpressing plants showed a late-flowering phenotype. These findings demonstrate that a type-A RR inhibits Ehd1 activity by binding to form an inactive complex.