The histone methyltransferase SDG724 mediates H3K36me2/3 deposition at MADS50 and RFT1 and promotes flowering in rice

Abstract

Chromatin modifications affect flowering time in the long-day plant Arabidopsis thaliana, but the role of histone methylation in flowering time regulation of rice (Oryza sativa), a short-day plant, remains to be elucidated. We identified a late-flowering long vegetative phase1 (lvp1) mutant in rice and used map-based cloning to reveal that lvp1 affects the SET domain group protein 724 (SDG724). SDG724 functions as a histone methyltransferase in vitro and contributes to a major fraction of global histone H3 lysine 36 (H3K36) methylation in vivo. Expression analyses of flowering time genes in wild-type and lvp1 mutants revealed that Early heading date1, but not Heading date1, are misregulated in lvp1 mutants. In addition, the double mutant of lvp1 with photoperiod sensitivity5 (se5) flowered later than the se5 single mutant, indicating that lvp1 delays flowering time irrespective of photoperiod. Chromatin immunoprecipitation assays showed that lvp1 had reduced levels of H3K36me2/3 at MADS50 and RFT1. This suggests that the divergent functions of paralogs RFT1 and Hd3a, and of MADS50 and MADS51, are in part due to differential H3K36me2/3 deposition, which also correlates with higher expression levels of MADS50 and RFT1 in flowering promotion in rice.

Figure 1.

Phenotype of the lvp1 Mutant. (A) Phenotypes of 110-d-old wild-type (WT; left) and lvp1 (right) mutant plants grown in a paddy field at Beijing. (B) Panicles of 150-d-old wild-type (left) and lvp1 plants (right) plants grown in Beijing. The lvp1 mutant was not heading at this time and only developed immature panicles. (C) Heading date investigation of wild-type and lvp1 plants grown at three places of different latitudes. Error bars indicate sd (n = 20).

Figure 2.

Map-Based Cloning of LVP1. (A) Primary mapping of LVP1 localized the gene to the region between the molecular markers S1 and S2 on chromosome 9. (B) Fine mapping of LVP1 using 1147 F2 progeny plants further delimited the gene to a 139-kb genomic region on chromosome 9. (C) Genomic structure of LVP1/SDG724. Black boxes show exons, white boxes show untranslated regions, and brown lines show introns. “G-C” and “115 bp Added” describe the mutation of the lvp1-1 allele; “79 bp Deleted” describes the mutation of the lvp1-2 allele. F and R indicate the primer pair used in (E) and are listed in Supplemental Table 1 online. (D) Allelism test between lvp1-1 and lvp1-2. WT, wild type. (E) Genotyping of SDG724 transcripts in wild-type, lvp1-1, and lvp1-2 plants by RT-PCR. The FR primer pairs are indicated in (C) and listed in Supplemental Table 1 online.

Figure 3.

SDG724 Acts as a SET Domain–Containing Histone Methyltransferase. (A) and (B) In vitro HMTase assay analyses. Calf thymus core histones (A) and oligonucleosomes (B) were used as substrates, and tritium-labeled SAM was used as methyl donor. Proteins analyzed for enzyme activity are pointed out with arrows. Top panel: Coomassie blue–stained protein gel. Bottom panel: Corresponding autoradiograph. (C) Analysis of the global levels of H3K36me1/2/3, H3K4me1/2/3, and H3K9me2 markers in wild-type (WT), lvp1-1, and lvp1-2 plants using H3K methylation tag-specific antibodies. Histone-enriched proteins were extracted from leaves of 50-d-old plants grown in Beijing under natural LD conditions. For each immunoblot, the same membrane was stripped and reblotted with an antibody against H3 as a loading control.

Figure 4.

Diurnal Expression Differences of Flowering Time Genes between Wild-type and lvp1 Plants Grown under LD Conditions. Leaf samples were collected every 4 h from 30-d-old plants grown under LD conditions. Each point represents at least three biological replicates. y axis, relative transcript levels of flowering time genes normalized to those of rice Ubiquitin. Error bars indicate sd (n = 3 or more). WT, wild-type; ZT, Zeitgeber time. Primers are listed in Supplemental Table 1 online.

Figure 5.

Diurnal Expression Differences of Flowering Time Genes between Wild-Type and lvp1 plants Grown under SD Conditions. Leaf samples were collected every 4 h from 25-d-old plants grown under SD conditions. Each point represents at least three biological replicates. y axis, relative transcript levels of flowering time genes normalized to those of rice Ubiquitin. Error bars indicate sd (n = 3 or more). WT, wild-type; ZT, Zeitgeber time. Primers are listed in Supplemental Table 1 online.

Figure 6.

H3K36me2/3 Levels Are Reduced at the MADS50 Locus in lvp1 Mutant Plants. ChIP samples were analyzed by quantitative PCR of six regions of MADS50 and five regions of Ehd1. The rice Actin1 gene was used as an internal control. The relative levels of specific histone H3 Lys methylation marks were calculated as the ratio of specific methylation marks over total H3 and normalized as the ratio over total H3 at Actin1; error bars indicate sd (n = 3 or more). Primers are listed in Supplemental Table 1 online. (A) Genomic structure of the MADS50 and Ehd1 loci. Regions assayed in (B) are indicated by lines and numbered. (B) ChIP analysis of H3K36me2/3 and H3K4me3 deposited on the chromatin of MADS50 and Ehd1 loci in wild-type (WT) and lvp1 mutant plants.

Figure 7.

H3K36me2/3 Levels Are Severely Reduced at the RFT1 Locus in lvp1 Mutant Plants. ChIP samples were analyzed by quantitative PCR of six different regions of each gene. The rice Actin1 gene was used as an internal control. The relative levels of specific histone H3 Lys methylation marks were calculated as the ratio of specific methylation marks over total H3 and normalized as the ratio over total H3 at Actin1; error bars indicate sd (n = 3 or more). Primers are listed in Supplemental Table 1 online. (A) Alignment of RFT1 and Hd3a protein sequences using ClustalX and GenDoc. Shades of black indicate conserved amino acids. (B) Genomic structure of the RFT1 and Hd3a loci on chromosome 6. Black boxes indicate exons, and white boxes indicate untranslated regions. Regions assayed in (C) are indicated by lines and numbered. (C) ChIP analysis quantifying H3K36me2/3 and H3K4me3 levels in the chromatin of the RFT1 and Hd3a loci in wild-type (WT) and lvp1 mutant plants.

Figure 8.

Proposed Model for the SDG724 Flowering Pathway in Rice. The black lines show previously identified pathways, and the gray lines indicate pathways added to this model from our study. SDG724 is required for high expression of MADS50, MADS51, Ehd1, RFT1, and Hd3a, but influences H3K36me2/3 levels only in MADS50 and RFT1 chromatin.