Homodimerization of Ehd1 Is Required to Induce Flowering in Rice

Abstract

In plants, flowering time is elaborately controlled by various environment factors. Ultimately, florigens such as FLOWERING LOCUS T (FT) or FT-like molecules induce flowering. In rice (Oryza sativa), Early heading date 1 (Ehd1) is a major inducer of florigen gene expression. Although Ehd1 is highly homologous to the type-B response regulator (RR) family in the cytokinin signaling pathway, its precise molecular mechanism is not well understood. In this study, we showed that the C-terminal portion of the protein containing the GARP DNA-binding (G) domain can promote flowering when overexpressed. We also observed that the N-terminal portion of Ehd1, carrying the receiver (R) domain, delays flowering by inhibiting endogenous Ehd1 activity. Ehd1 protein forms a homomer via a 16-amino acid region in the inter domain between R and G. From the site-directed mutagenesis analyses, we demonstrated that phosphorylation of the Asp-63 residue within the R domain induces the homomerization of Ehd1, which is crucial for Ehd1 activity. A type-A RR, OsRR1, physically interacts with Ehd1 to form a heterodimer. In addition, OsRR1-overexpressing plants show a late-flowering phenotype. Based on these observations, we conclude that OsRR1 inhibits Ehd1 activity by binding to form an inactive complex.

Figure 1.

Analyses of transactivation of Ehd1. A, Schematic diagrams of Ehd1 and its truncated molecules used for assay of transactivation. Numbers represent positions of amino acids. R, receiver domain; I, inter domain; G, GARP DNA-binding domain; C, C-terminal region. B, Analyses of transactivation. Full-length or truncated Ehd1 cDNA was inserted into pBD_GAL4 Cam vector containing GAL4 DNA-binding domain, then transferred into yeast cells. Empty vector (pBD_empty) and SNB_1 ∼ 275 were used as negative and positive control, respectively. Values calculated for β-galactosidase activity are averages of three independent experiments. Bars represent standard deviations.

Figure 2.

Phenotypes of transgenic plants over-expressing full-length or truncated Ehd1. A, Schematic diagrams of full-length and truncated forms of Ehd1 used for generation of transgenic plants. Top, full-length protein; middle, truncated Ehd1 with IGC domain; bottom, truncated Ehd1 with RI domain. B, Phenotypes of transgenic plants over-expressing full-length Ehd1 (Ehd1 OX) and truncated Ehd1 (RI OX and IGC OX) grown under SD conditions. Arrows indicate bolted panicles. Scale bar = 10 cm. C, Heading dates under SD conditions. Two independently transformed plants that expressed introduced gene at high levels were analyzed. Date were recorded when first panicle bolted. Heading dates of wild-type plants and transgenics over-expressing full-length Ehd1 are indicated as dashed lines. Error bars show standard deviations. n = 8 or more. WT, wild type.

Figure 3.

Interaction analyses of Ehd1 and truncated Ehd1. A, Homerization analysis of Ehd1. coIP analyses performed between Ehd1-HA and Ehd-Myc. B, Homerization analysis of truncated Ehd1 containing R and I regions. coIP analyses were performed between Ehd1 RI fragment tagged with HA (RI-HA) and Ehd1 RI fragment tagged with Myc (RI-Myc). C, Interaction analyses among full-length Ehd1 and truncated Ehd1. Truncated Ehd1 containing I, G, and C regions was tagged with HA (IGC-HA) or Myc (IGC-Myc). Homomerization of fragment as well as interaction with RI fragment or full-length Ehd1 were examined. Tagged proteins were expressed in rice suspension cells after transformation of tagged constructs. Expressed proteins were immunoprecipitated with anti-Myc antibodies or anti-HA antibodies, then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Signals were detected by HRP-conjugated anti-HA antibodies or anti-Myc antibodies. Inclusion or exclusion of construct is shown by plus or minus. D, Heading date of SD-grown transgenic plants over-expressing truncated Ehd1 with R domain (R OX) or GC domain (GC OX). Error bars indicate standard deviations. n = 8 or more. Scale bar = 10 cm. WT, wild type. E, Y2H assays of interaction between Ehd1 RI fragment (BD-RI) with deletion derivatives of I region. AD-empty and BD-RI was used as a negative control.

Figure 4.

Flowering phenotypes of transgenic plants over-expressing Asp-63 mutants. A, Point mutations and aa substitutions in 63^rd Asp residue of Ehd1. B, Phenotypes of SD-grown transgenic plants expressing mutated Ehd1. Bar = 10 cm. C and D, Heading date of transgenic plants expressing mutated Ehd1 grown under SD (C) or LD (D) conditions. Two independently transformed plants that expressed introduced gene wild-type plants and transgenics over-expressing wild-type Ehd1 are indicated as dashed lines. Error bars show standard deviations. n = 6 or more. E and G, Quantitative real-time PCR analyses of Ehd1 (E), Hd3a (F), and RFT1 (G) expression in plants over-expressing mutated Ehd1. Leaf blades were harvested at ZT 4 h from 21-DAG plants grown under SD conditions. The y axis shows the relative transcript level compared with rice Ubi1 expression. Transcript levels for florigen genes in transgenic plants over-expressing wild-type Ehd1 are indicated as dashed lines. Scale bar = 10 cm. Error bars show standard deviations. n = 4 or more.

Figure 5.

Y2H assays for homomerization of mutated Ehd1. Homomerization analyses by Y2H experiments. Wild-type Ehd1 and Asp-63 mutants were inserted into pGAD424 or pBD_GAL4 cam vector, and their interaction was analyzed by testing growth on minimal medium. Transformants were grown in liquid medium without Leu and Trp to achieve optical density A₆₀₀ = 1. Afterward, 10 μL of undiluted or diluted yeast cell suspension was dropped onto minimal medium plates supplemented with 5 mm 3-amino-1,2,4-triazole but without Leu, Trp, and His, and held for 3 d. Values for relative β-galactosidase activity are averages from 3 independent assays. Error bars indicate standard deviations.

Figure 6.

Phosphorylation-dependent homomerization of Ehd1. co-IP analyses were performed between Ehd1-HA and Ehd1-Myc after CIP treatment. Vectors were transformed into protoplasts prepared from Oc cells. Before immunoprecipitation with anti-HA antibodies, the protein extracts were incubated with or without CIP for 2 h at 37°C. Immunoprecipitated proteins were separated by SDS-PAGE, and signals were detected by HRP-conjugated anti-Myc antibodies.

Figure 7.

Characterization of OsRR1. A, Interaction analyses between Ehd1 and type-A RRs of rice by co-IP experiments. Full-length cDNAs of Ehd1 and rice type-A RRs were tagged with Myc and placed under control of Ubi1 promoter. Constructs, together with Ehd1-HA tag molecule, were introduced into protoplasts prepared from Oc cells, and expressed proteins were immuno-precipitated with anti-HA antibody. After proteins were separated via SDS-PAGE, signals were detected by HRP-conjugated anti-Myc antibody. B, Levels of OsRR1 transcripts in over-expressing plants. Two independently transformed plants that expressed introduced gene at high levels were analyzed. C, Heading dates for OsRR1 OX transgenic plants and wild-type controls grown under SD conditions. Bar = 10 cm. Error bars indicate standard deviations. n = 6 or more.

Figure 8.

A putative model for flowering inhibition by OsRR1. Ehd1 is expressed during period of flowering induction. Ehd1 protein is activated by phosphorylation of middle D in R domain. Phosphorylation induces dimerization of Ehd1 by structural changes, which stimulates transcription of target genes Hd3a and RFT1. OsRR1 binds to Ehd1 to interfere with homomerization of Ehd1. This results in suppression of target gene expression, thereby delaying flowering.