Ehd4 encodes a novel and Oryza-genus-specific regulator of photoperiodic flowering in rice

Abstract

Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (Oryza sativa L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the CO-FT module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (Hd1-Hd3a in rice). However, in rice, there is a unique Ehd1-dependent flowering pathway that is Hd1-independent. Here, we report isolation and characterization of a positive regulator of Ehd1, Early heading date 4 (Ehd4). ehd4 mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that Ehd4 encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids in vitro and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. Ehd4 expression is most active in young leaves with a diurnal expression pattern similar to that of Ehd1 under both short-day and long-day conditions. We show that Ehd4 up-regulates the expression of the "florigen" genes Hd3a and RFT1 through Ehd1, but it acts independently of other known Ehd1 regulators. Strikingly, Ehd4 is highly conserved in the Oryza genus including wild and cultivated rice, but has no homologs in other species, suggesting that Ehd4 is originated along with the diversification of the Oryza genus from the grass family during evolution. We conclude that Ehd4 is a novel Oryza-genus-specific regulator of Ehd1, and it plays an essential role in photoperiodic control of flowering time in rice.

Figure 1. 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.

Figure 2. Map-based cloning of Ehd4.

(A) Location of the Ehd4 locus on rice chromosome 3. (B) High-resolution linkage map of Ehd4. (C) Candidate genes on BAC OSJNBb0005F16. (D) Structure of the Ehd4 gene. Lines, black and white boxes represent introns, exons and untranslated regions, respectively. The base change from G to A creates an early stop codon (Asterisk). (E) Complementation of ehd4. Ehd4 was driven by either the native promoter (pEhd4::Ehd4) or the maize Ubiquitin-1 promoter (pUbi::Ehd4). T2 plants of two pEhd4::Ehd4 lines (#26 and #34) and two pUbi::Ehd4 lines (#18 and #24) were measured (n = 10). All plants were grown under both SD and LD conditions.

Figure 3. 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.

Figure 4. 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 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.

Figure 5. The rhythmic expression pattern of Hd3a, RFT1, and Ehd1, but not Hd1, was abolished in the ehd4 mutant plants under both SDs and LDs.

SDs (A, C, E and G); LDs (B, D, F and H). The open and filled bars at the bottom represent the light and dark periods, respectively. The rice Ubiquitin-1 (UBQ) gene was used as the internal control. Values are shown as mean±s.d. (standard deviations) of three independent experiments and two biological replicates.

Figure 6. Quantitative RT–PCR analysis of representative flowering-related genes and Ehd4 in various flowering-time mutants or their NILs (near-isogenic lines) and corresponding WTs under SDs and LDs.

(A) Transcript level of Ehd2, Ehd3, OsMADS50, OsGI, OsMADS51, OsphyB, OsCOL4 and DTH8 in WT (Kita-ake) and ehd4 plants. (B) Transcript level of Ghd7, Hd3a, RFT1, Ehd1 and Hd1 in WT (Nipponbare) and ehd4-Nip plants. (C) Transcript level of Ehd4 in various flowering-time mutants or their NILs (near-isogenic lines) and corresponding WTs. Dongjin and the osphyb mutant ; Tohoku IL9 and the ehd2 mutant ; Tohoku IL9 and the ehd3 mutant ; Dongjin and the osmads50 mutant ; Dongjin and the osmads51 mutant ; Nipponbare and a NIL carrying a non-functional Hd1 allele ; Asominori and a NIL carrying a nonfunctional DTH8 allele ; A NIL carrying a functional Ghd7 allele and a NIL carrying a non-functional in the Shanyou 63 background . Taichun 65 carrying a non-functional Ehd1 allele and a NIL carrying a functional Ehd1 allele ; Nipponbare carrying a partially functional Hd3a allele and a NIL carrying a functional Hd3a allele ; Penultimate leaves were harvested around reported peak expression level of each gene during the 24 hrs photoperiod - at dawn for OsphyB, OsCOL4, Ehd1, Ehd2, Hd3a, RFT1 and Ehd4, 3 h after dawn for Ghd7, 8 h after dawn for Ehd3, OsMADS50, OsMADS51 and DTH8 and immediately after dusk for OsGI and Hd1 from 28 d-old (SDs) and 35 d-old (LDs) plants. The rice Ubiquitin-1 (UBQ) gene was used as the internal control. Values are shown as mean±s.d (standard deviations) of three independent experiments and two biological replicates.

Figure 7. Complementation of ehd4 by over-expression of Ehd1.

(A) Phenotypes of the mutant and transgenic plants at heading stage. (B) Ehd1, when driven by the maize Ubiquitin-1 promoter (pUbi::Ehd1), was able to rescue the flowering phenotype of ehd4 (T0 plants, n = 8). All plants were grown under SDs.

Figure 8. Natural variations in the Ehd4 coding region among rice germplasm core collection.

(A) Haplotype network of the Ehd4 alleles in 86 rice accessions. Haplotype frequencies are proportional to the area of the circles. The proportion of wild rice and two cultivated subgroups (indica and japonica) in each haplotype is represented by different colors. (B) The polymorphic nucleotides of Hap_2 and Hap_3 of Ehd4 gene in the core collection. The number on the top shows the position of nucleotide polymorphisms in the coding region starting from the ATG start codon. (C) Geographic distribution of the cultivated rice accessions belonging to Hap_2 and Hap_3. (D) Flowering time of transgenic plants carrying two major haplotypes of Ehd4 driven by the maize Ubiquitin-1 promoter in ehd4 (Kita-ake background) and NIL carrying Ehd4 ^Hap3 compared with the 93-11 parental plants. T2 plants of two pUbi::Ehd4 ^Hap3 (#18 and #24) and two pUbi::Ehd4 ^Hap2 (#12 and #16) lines were measured (n = 15). All plants were grown in the natural long day field conditions. Values are means±s.d. (standard deviations) (n = 15). **Significant at 1% level; n.s., not significant.