LC2 and OsVIL2 Promote Rice Flowering by Photoperoid-Induced Epigenetic Silencing of OsLF  Jun Wang, Jiang Hu, Qian Qian, Hong-Wei Xue  Molecular Plant  Volume 6, Issue 2, Pages 514-527 (March 2013) DOI: 10.1093/mp/sss096 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Rice LC2 Interacts with OsVIL2. (A) Phylogenetic tree of VIL proteins in plants. (B) In vivo interaction of LC2 and OsVIL2 in N. benthamiana leaves. Coding regions of LC2 and OsVIL2 are fused into pCAMBIA1300–nLUC and pCAMBIA1300–cLUC, respectively, and used for leaf dot infiltration. Agrobacterium strains expressing LC2–nLuc and cLuc–RAR1 or cLuc–OsVIL2 and SGT1a–nLuc were used as negative controls; strains expressing SGT1a–nLuc and cLuc–RAR1 were used as positive controls. Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 LC2 and OsVIL2 Are Positive Rice Flowering Promoters. (A) lc2 and OsVIL2–RNAi plants displayed delayed flowering time under SD. Plants were planted at different photoperiods and heading dates were calculated when the first panicle was bolted. Error bars indicate SD (n≥8). (B) qRT–PCR analysis confirmed the suppressed expression of Hd1, Hd3a, and Ehd1 in lc2 and OsVIL2–RNAi plants, and recovered expression of Hd1, Hd3a, and Ehd1 in lc2 plants with complementary expression of LC2. qRT–PCR analysis was performed using RNAs extracted from leaf blades when the control plants entered into jointing stage (all the materials were sampled 6h after transition from dark to light in the same day). Relative transcript levels were calculated by comparing with those of rice ACTIN. Error bars indicate standard deviations (SD, n=3). All experiments were repeated at least twice, with similar results. Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Expressions of LC2 and OsVIL2 Are Induced by SD. (A) RNA in situ hybridization analysis of LC2 and OsVIL2. According to the diurnal expression patterns of LC2 and OsVIL2, materials were harvested at ZT 6 under SD conditions. Leaf blades of ZH11 at 30DAG were cross-sectioned and hybridized with LC2 and OsVIL2-specific antisense or sense probes. Red arrows indicate the mesophyll cells. Bar=100μm. (B) qRT–PCR analysis showed that LC2 and OsVIL2 display the similar rhythmically diurnal expression pattern. Leaf blades of ZH11 at 45DAG were harvested every 3h starting from 9:00am. The black rectangle represents dark and the red rectangle represents light. For (B) and (C), relative transcript levels were calculated by comparing with those of rice ACTIN. Error bars indicate SD (n=3) and all experiments were repeated at least twice, with similar results. (C) The mRNA levels of LC2 and OsVIL2 are induced by SD treatment. ZH11 seedlings at 5DAG were divided into two replicates, which were treated by LD (14-h light/10-h dark) and SD (10-h light/14-h dark), respectively, for 10d. Entire seedlings were harvested at 24 time points throughout a whole day for mRNA extraction. Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Unaffected Expression of Genes Involved in Light Perception, as Well as the Diurnal Rhythm of OsGI in lc2. (A) qRT–PCR analysis of expression of light receptors (PHYA, B, and C; CRY1 and 2) in lc2 and YUNDAO32. Leaf blades of 45DAG were harvested for RNA extraction. Relative transcript levels were calculated by comparing with those of rice ACTIN. Error bars indicate SD (n=3). All experiments were repeated at least twice, with similar results. (B) qRT–PCR analysis of expression of OsGI and Hd1 in lc2 and YUNDAO32 revealed the unaltered diurnal rhythm of OsGI in lc2. Leaf blades of 45DAG lc2 and YUNDAO32 were harvested every 3h for 36h. The black rectangle represents dark and the red rectangle represents light. Relative transcript levels of OsGI and Hd1 were calculated by comparing with those of rice ACTIN. Error bars indicate SD (n=3). All experiments were repeated at least twice, with similar results. Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 OsLF Is Epigenetically Silenced by LC2. (A) qRT–PCR analysis revealed the enhanced expression of OsLF, OsMADS56, OsCO3, and OsCOL4 in lc2. qRT–PCR analysis was performed using RNAs extracted from developing leaf blades (all the materials were sampled on the same day; plants were grown under SD conditions, 12-hlight/12-h dark). Relative transcript levels were calculated by comparing with those of rice ACTIN. Error bars indicate SD (n=3). All experiments were repeated at least twice, with similar results. (B) Chromatin immunoprecipitation analysis revealed the reduced H3K27 tri-methylation level in the promoter of OsLF and the unreduced H3K27 tri-methylation in the promoter of OsMADS56, OsCO3, and OsCOL4 in lc2. For OsMADS56, OsCO3, and OsCOL4, four pairs of primers covering the 1-kb promoter region were used and only one representative result is shown. For the OsLF locus diagram, OsLF-EMSA is the same DNA fragment which could be bound by the LC2 protein in (C); OsLF-ChIP-1 is a 206-bp fragment which lies in between the translational start (ATG) and OsLF-EMSA; OsLF-ChIP-2 is a coding region adjacent to the translational start (ATG). Developing leaf blades were harvested for formalin fixation. Immunoprecipitated DNA was amplified by qRT–PCR and enrichment was represented as percentage of Input (% Input). All experiments were repeated at least twice, with similar results. (C) Chromatin immunoprecipitation analysis revealed that SD induced the H3K27 tri-methylation level in the promoter of OsLF. ZH11 seedlings were divided into two replicates, which were treated by LD (14-h light/10-h dark) and SD (10-h light/14-h dark), respectively, for 10d. Entire seedlings were harvested for formalin fixation. Immunoprecipitated DNA was amplified by qRT–PCR and enrichment was represented as percentage of Input (% Input). Experiments were repeated twice, with similar results. (D) EMSA results showed that LC2 could bind to the promoter region of OsLF. Each EMSA competitive binding analysis was performed with increasing amounts of unlabeled probe (competitor). The concentration of unlabeled probe was 10, 100, and 200 times that of the labeled probe, respectively. The fragment (amplified by using Primers MADS56-1S and MADS56-1A) is used as a non-related sequence (NRS). Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 OsLF Directly Binds to the Promoter Region of Hd1. (A) Presence of six regions containing bHLH cis-elements within the 3-kb promoter region of Hd1. The typical bHLH cis-element is CANNTG. (B) OsLF directly binds to three Hd1 promoter regions containing the CANNTG motif. Each EMSA competitive binding analysis was performed with increasing amounts of unlabeled probe (competitor). The concentration of unlabeled probe was 10, 100, and 200 times of that of the labeled probe, respectively. (C) Substitution analysis confirmed the direct binding of OsLF to the Hd1 promoter regions containing the CANNTG motif, Hd1 promoter-3 (upper panel), and Hd1 promoter-5 (lower panel). For Δprobe, ACAACA was substituted for the CAGGTG motif in Hd1 promoter-3; ACAACA and ATCCCC were substituted for CAGATG and CAGTTG motifs in Hd1 promoter-5. The protein amount used in lane 2 was one-third of that of lane 3; the concentration of unlabeled probe used in lane 4 was 50 times the labeled probe. (D) The substituted cold competitors lost the ability to compete with Dig-labeled probes. The concentration of the unlabeled substituted probe was 100 and 200 times that of the labeled probe, respectively. Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Comparison of the PRC2 Function Model between Arabidopsis and Rice. (A) In Arabidopsis, VIN3 and VRN5 are induced by cold and interact with each other. Other components are then recruited to form a full PRC2 complex, which represses the FLC expression through mediating the H3K27 tri-methylation modification in the FLC locus. The inhibition of FT is thus removed and the plant begins to flower. (B) In rice, LC2 and OsVIL2 are induced by SD and interact with each other and the LC2-OsVIL2 hetero-dimer directly binds to the promoter of OsLF. Other components are then recruited to form a full PRC2 complex, which represses the OsLF expression through mediating the H3K27 tri-methylation modification in the OsLF promoter. The inhibition of Hd1 is then removed to stimulate the expression of flowering-stimulated genes, which eventually promotes rice flowering. Molecular Plant 2013 6, 514-527DOI: (10.1093/mp/sss096) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions