Os03g0112700

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Annotated Information

Function

   This gene is called Early heading date 4 (Ehd4), and it is about the flowering.Ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Ehd4 may function by coding a transcriptional regulator. The transcriptional regulator can also bind to the DNA or RNA when it regulates the expression of gene. Some experiments indicate that there is an activation domain located in the middle region near the C-terminal of EHD4, it can also bind to the DNA or RNA. In addition to Ehd2 and Ehd3,Ehd4 can also promote  the expression of Ehd1 by a different pathway.The expression of Ehd1 by a different pathway. It can promotes the expression of Hd3a and Rft1 by a different pathways though Ehd1.Rice has a unique, Hd1-independent flowering pathway that is mediated by Early heading date 1 (Ehd1). Ehd1 encodes a B-type response regulator that is highly conserved in cultivated rice, but has no homolog in Arabidopsis. It has been shown that Ehd1 positively regulates the expression of Hd3a and RICE FLOWERING LOCUS T1, the closest paralog of Hd3a that works as a LD ‘florigen’. Circumstantial evidence suggests that Ehd1 is a critical convergence point of regulation by multiple signaling pathways.

EHD4 may act as a transcriptional regulator In higher plants, CCCH-type zinc finger proteins have been shown to regulate gene expression by binding to DNA or RNA molecules in the nucleus. We fused Ehd4 with GFP and transiently expressed the EHD4-GFP fusion protein in rice leaf protoplasts. EHD4-GFP was exclusively co-localized with the OsMADS3-mCherry fusion protein (a nuclear marker) in the nucleus (Figure 1A–1C), indicating that EHD4 functions in the nucleus. We further fused EHD4 and its various deletions with the GAL4 DNA binding domain and investigated if EHD4 has transcriptional activation activity in yeast. Full-length wild type EHD4 and an EHD4 variant with only the CCCH motif removed were able to activate the reporter gene expression (Figure 1D). Further deletion of the C terminal region resulted in a dramatic reduction of the activation activity, whereas deletion of both the N-terminal and CCCH motif only had mild effects (Figure 1D). These observations suggest that the activation domain is located in the middle region close to the C-terminal of EHD4. In addition, a nucleic acid binding assay demonstrated that the C-terminal region, but not the N-terminal region, can bind to both double- and single-stranded calf thymus DNA and ribohomopolymers in vitro, and that removal of the CCCH motif from the C-terminal abolished the binding activity (Figure 1E). These results strongly support the notion that EHD4 likely functions as a transcriptional activator and that the CCCH motif is essential for its nucleic acid binding activity.

Figure1. EHD4 is a nuclear protein with intrinsic transcriptional activation and nucleic acid binding activities. (A) Sub-cellular localization of EHD4-GFP fusion protein. (B) The nuclear marker MADS3-mCherry fusion protein. (C) Merged image of (A) and (B) under bright field. Scale bar = 10 µm in (A) to (C). (D) Transactivation assays of EHD4 and its deletion derivatives in the yeast GAL4 system. Full length EHD4 and several deletion derivatives of EHD4 (pEhd4-Δ, pEhd4-N and pEhd4-CΔ) were used in assays. The empty vector (BD-MCS) and BD-DST [56] were used as negative and positive control, respectively. Transformants were dropped onto SD/Trp- and SD/His- plates to allow growth of 48 hours before taking pictures. Values in β-galactosidase activity are means of three independent experiments. Bars stand for standard deviations. BD, DNA-binding domain of GAL4. (E) The CCCH motif is essential for binding to nucleic acids. C terminal, N terminal or C terminal without CCCH motif of EHD4 was expressed in E.coli and purified for binding assays. Deletion of the CCCH motif abolished the binding to ribohomopolymers and both double- and single-stranded calf thymus DNA.

Ehd4 regulates expression of the “florigen” genes through Ehd1. Ehd4 functions upstream of Ehd1, but largely independent of other known regulators of Ehd1. Consistent with this, down regulation of Ehd1, Hd3a and RFT1 in ehd4 was also seen in the Nipponbare background and constantly seen at different stages during plant development.

Expression

   Ehd4 most actively express in young leaves. The diurnal expression pattern is similar to that of Ehd1 under both short-day and long-day conditions.Ehd encodes a new CCCH-type zinc finger protein,which located in to the nucleus,and it can bind the nucleic acids in the nucleus.Through the qRT-PCR,It showed that it accumulates after dusk, reaching a peak at dawn, and damping rapidly thereafter under both SDs and LDs.

Ehd1 expression is promoted by a number of positive regulators. Among them, OsMADS51 encodes a type I MADS-box protein and induces Ehd1 expression under SDs, whereas a rice homolog of Arabidopsis SOC1 (Suppressor of Overexpression of Constant1), OsMADS50, was identified as a promoter of Ehd1 expression under LDs. Recently, it was shown that Ehd1 expression could be independently up-regulated by Early heading date 2/Rice Indeterminate 1/Oryza sativa Indeterminate 1 (referred to as Ehd2 hereafter) and Early heading date 3 (Ehd3) under both SDs and LDs Expression of Ehd4 is constitutive and diurnal We examined the expression levels of Ehd4 in various tissues and at different stages of leaf development (Figure 2A) by using qRT-PCR. Ehd4 transcripts were detected in all tissues examined, but the highest expression was found in emerging young leaves and the lowest level in fully expanded leaves (Figure 2B). Histochemical staining of transgenic plants carrying the GUS reporter gene driven by the Ehd4 promoter indicated that GUS was expressed in all tissues examined and was most abundant in the vascular tissue and apical meristem (Figure 2C–2I). The expression of Ehd4 showed a diurnal expression pattern in leaves. It accumulates after dusk, reaching a peak at dawn, and damping rapidly thereafter under both SDs and LDs (Figure 2J). Moreover, Ehd4 was expressed constantly during the vegetative growth from the second week to the 10th week after germination (Figure 2K).

Figure2. Expression pattern of Ehd4. (A) 30-d old wild-type plants (Kita-ake) grown under SDs were used for quantitative RT-PCR. DL1, newly emerging leaf; DL2, expending leaf; DL3, fully expended leaf; ASA, around the shoot apex. (B) Ehd4 transcript levels in various organs (means±s.d, n = 3). (C) to (I) GUS staining of various organs in pEHD4::GUS transgenic plants. (C) Root; (D) Floret; (E) Stem; (F) to (H) Transverse sections of stem, immature leaf and sheath, respectively; (I) Longitudinal section of the shoot apical meristem (SAM). Arrow indicates phloem in (F) and (G) and SAM in (I). (J) and (K) Rhythmic and developmental expression of Ehd4. The rice Ubiquitin-1 (UBQ) gene was used as the internal control. Values are shown as mean±s.d of three independent experiments and two biological replicates. The open and filled bars at the bottom represent the light and dark periods, respectively. s.d: standard deviations. doi:10.1371/journal.pgen.1003281.g003

Evolution

Ehd4 is likely a single gene,it's unique and conserved in rice,after analysis about Ehd4' sequence of many rices.

mutation

Ehd4 mutants showed a never flowering phenotype under natural long-day conditions. The ehd4 mutant was initially identified from a tissue culture-derived population of rice cv Kita-ake (japonica) under natural-day conditions in a paddy field in Beijing (39°54′N, 116°23′E), China (2006). In an effort to isolate genes that are essential for promoting flowering time in rice, we generated a large T-DNA population in a day-length neutral, early flowering variety Kita-ake (O. sativa ssp. japonica). Kita-ake (Kit) has been widely used in rice transformation experiments because of its short life cycle. Kit flowers about two months after germination under both SDs (10 h light/14 h dark) and LDs (14.5 h light/9.5 h dark) conditions in the controlled growth chamber, as well as under natural long-day field conditions (NLDs) in Beijing (39°54′N, 116°23′E), North China (Figure 3A and 3B). To understand the day-length neutral nature of Kita-ake, we cloned ten genes reported to have significant effect on flowering time in rice, including seven genes that promote flowering (Ehd 1 to 3, OsMADS50, OsMADS51, Hd3a and RFT1) and three genes that suppress flowering under LDs (Hd6, Hd1 and Ghd7), and compared them with the corresponding genes in Nipponbare (Nip), a japonica variety that is sensitive to day-length.Compared with WT, ehd4 delayed flowering time by 49 d and 106 d under SDs and LDs, respectively (Figure 3B). Consistent with field observations, flowering time of the heterozygotes was also delayed under both SDs and LDs (Figure 3B). Notably, ehd4 had a similar leaf emergence rate to WT under both SDs and LDs (Figure 3C), indicating that the late flowering phenotype is not caused by retardation in growth rate. The mature ehd4 plants were taller, producing more but smaller seeds. The fertility of ehd4 plants was similar to that of WT (Figure 3D–3H).

Figure3. Characterization of Ehd4. (A) Never-flowering phenotype of ehd4 mutants in field (Top). WT, Kita-ake wild-type plants (Bottom). (B) Flowering time of ehd4, heterozygote (HETE) and WT plants under different day length conditions in Kita-ake (day-length neutral) and Nipponbare (day-length sensitive) backgrounds (n = 12). ND, natural-day; SD, short-day; LD, long-day. (C) ehd4 plants had the same leaf emergence rate as WT (Kita-ake) under both SDs and LDs (n = 8). Arrow indicates the flowering time of WT plants. (D) Panicle morphology of WT and ehd4 plants. (E) to (H) Comparisons of grain number per panicle (E), 1000-grain weight (F), plant height (G) and fertility (H) between WT and ehd4 plants. Values are means±s.d. (standard deviations) (n = 15). **Significant at 1% level; n.s., not significant.

Labs working on this gene

National Key Laboratory for Crop Genetics and Germplasm Enhancement,Jiangsu Plant Gene Engineering Research Center,Nanjing Agricultural University,Nanjing,China National Key Facility for Crop Gene Resources and Genetic Improvement,Insitute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing,China Peking University,China

References

Gao H, Zheng X M, Fei G, et al. Ehd4 encodes a novel and Oryza-genus-specific regulator of photoperiodic flowering in rice[J]. PLoS genetics, 2013, 9(2): e1003281.