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, 41 (7), 665-675

Histone Deacetylase 701 (HDT701) Induces Flowering in Rice by Modulating Expression of OsIDS1

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Histone Deacetylase 701 (HDT701) Induces Flowering in Rice by Modulating Expression of OsIDS1

Lae-Hyeon Cho et al. Mol Cells.

Abstract

Rice is a facultative short-day (SD) plant in which flowering is induced under SD conditions or by other environmental factors and internal genetic programs. Overexpression of Histone Deacetylase 701 (HDT701) accelerates flowering in hybrid rice. In this study, mutants defective in HDT701 flowered late under both SD and long-day conditions. Expression levels of florigens Heading date 3a (Hd3a) and Rice Flowering Locus T1 (RFT1), and their immediate upstream floral activator Early heading date 1 (Ehd1), were significantly decreased in the hdt701 mutants, indicating that HDT701 functions upstream of Ehd1 in controlling flowering time. Transcript levels of OsINDETERMINATE SPIKELET 1 (OsIDS1), an upstream repressor of Ehd1, were significantly increased in the mutants while those of OsGI and Hd1 were reduced. Chromatin-immunoprecipitation assays revealed that HDT701 directly binds to the promoter region of OsIDS1. These results suggest that HDT701 induces flowering by suppressing OsIDS1.

Keywords: Hd1; OsGI; OsIDS1; flowering time; histone deacetylase.

Figures

Fig. 1
Fig. 1. Schematic diagram of HDT701 structure and comparison of flowering times between WT and hdt701-1 mutants
(A) Gene structure of HDT701. Black boxes indicate exons in coding region; lines connecting boxes, introns; gray box, 5′-UTR region; open box, 3′-UTR region. T-DNA is inserted into first intron of HDT701 in Line 1B-05907. Direction of promoterless GUS reporter gene is indicated within T-DNA (triangle). Primers F, R, and NGUS1 (marked with arrows) were used for genotyping. Scale bar, 500 bp. (B) HDT701 transcript levels in WT and hdt701-1 measured by RT-PCR. (C) Phenotypes of hdt701-1 and WT at heading stage under paddy field conditions. Scale bar, 10 cm. (D) Days to heading for WT and hdt701-1 plants under SD, LD, and field conditions. DAG, days after germination. Error bar indicate standard deviation; n = 8. Levels of significant difference are indicated by *** (P < 0.005). (E) GUS-staining of leaf blade cross-section from Line 1B-05907. (F) Close-up of leaf section at vasculature region. b, bulliform cells; e, epidermis; m, mesophyll cells; p, phloem; pp. phloem parenchyma; x, xylem. Scale bars, 50 μm (E) and 20 μm (F).
Fig. 2
Fig. 2. Generation of additional hdt701 alleles by CRISPR/Cas9 method
(A) Schematic diagram of HDT701 structure and sequence alignment of sgRNA target region displaying altered bases in mutant lines. The target sequence is underlined. Altered DNA sequences are indicated in red. (B) Phenotypes of hdt701 KO lines at heading stage. Scale bar, 10 cm. (C) Days to heading for WT, and KO Lines #1, 4, and 5 under natural paddy field conditions. Days to heading was scored when first panicle bolted. Error bars indicate standard deviations; n = 10. Levels of significant difference are indicated by ** (P < 0.01) and *** (P < 0.005).
Fig. 3
Fig. 3. Diurnal expression patterns of floral regulators in leaf blades of WT and hdt701-1 plants at 49 DAG under LD
Quantitative RT-PCR analyses of HDT701 (A), Hd3a (B), RFT1 (C), Ehd1 (D), OsGI (E), Hd1 (F), OsIDS1 (G), SNB (H), OsCOL4 (I), OsLFL1 (J), OsLF (K), and OsPhyB (L). Closed circles, WT; open circles, hdt701-1. y-axis, relative transcript level of each gene compared with that of rice Ubi. Error bars indicate standard deviations; n = 4 (technical replicates). Levels of significant difference are indicated by * (P < 0.05), ** (P < 0.01), and *** (P < 0.005).
Fig. 4
Fig. 4. Diurnal expression patterns of floral regulators in leaf blades of WT and hdt701-1 plants at 28 DAG under SD
Quantitative RT-PCR analyses of HDT701 (A), Hd3a (B), RFT1 (C), Ehd1 (D), OsGI (E), Hd1 (F), OsIDS1 (G), SNB (H), OsCOL4 (I), OsLFL1 (J), OsLF (K), and OsPhyB (L). Closed circles, WT; open circles, hdt701-1. y-axis, relative transcript level of each gene compared with that of rice Ubi. Error bars indicate standard deviations; n = 4 (technical replicates). Levels of significant difference are indicated by * (P < 0.05), ** (P < 0.01), and *** (P <0.005).
Fig. 5
Fig. 5. Chromatin-immunoprecipitation (ChIP) assays of OsIDS1 chromatin and SNB chromatin
(A) Genomic structures of OsIDS1 and SNB. Tested areas are numbered. (B) ChIP assay of HDT701 enrichment on OsIDS1 chromatin, using HDT701-HA-tagged transgenic plants. Transgenic plants expressing HA tag alone served as control. Leaf blades were harvested at 30 DAG under SD. Percent-of-input method was used for normalization. (C) ChIP assay of HDT701-HA enrichment on SNB chromatin, as described in (B). Levels of significant difference are indicated by ** (P < 0.01). Error bars show standard deviations, n = 3. The entire experiment was conducted two times.
Fig. 6
Fig. 6. Expression levels of HDT701 in osphyb (A), oscol4 (B), osgi (C), and hd1 (D)
Total RNAs were prepared from leaf blades at 42 DAG under LD. Error bars display standard deviations; n = 4 (technical replicates).
Fig. 7
Fig. 7
Model for regulatory pathway governed by HDT701 for control of flowering time.

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