Volume 10, Issue 7, Pages 948-961 (July 2017) The DTH8-Hd1 Module Mediates Day-Length-Dependent Regulation of Rice Flowering  Anping Du, Wei Tian, Menghao Wei, Wei Yan, Hang He, Da Zhou, Xi Huang, Shigui Li, Xinhao Ouyang  Molecular Plant  Volume 10, Issue 7, Pages 948-961 (July 2017) DOI: 10.1016/j.molp.2017.05.006 Copyright © 2017 The Author Terms and Conditions

Figure 1 93-11 Contains an Hd1 Allele with a Mutation in the CCT Domain-Encoding Region. (A) Flowering phenotypes of 93-11 (left) and CSSLHd1(Nip) (right). Plants were grown under natural long-day conditions (LD). Photograph was taken when 93-11 flowered. (B) Frequency distribution for flowering time in F2 population of CSSLHd1(Nip) and 93-11. Plants were grown under natural LD. Chi-square test results check the segregation ratio of flowering phenotypes in F2. (C) The red arrowhead denotes a 4-bp deletion (AAGA) in the 93-11 Hd1 allele compared with the Nipponbare Hd1 allele. This deletion causes a mutation in the CCT domain of 93-11 Hd1 protein. Protein types are denoted as in a previous report (Takahashi et al., 2009). The markers for fine mapping are in black, and the InDel-1 marker in Hd1 is in red. (D) Expression of Ehd1 and Hd3a in 93-11 and CSSLHd1(Nip) in natural LD as indicated by real-time qPCR results. Data are shown as means ± standard deviation; n = 3. Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 2 Suhui527 Contains a Non-functional dth8 Allele. (A) Phenotypes of 93-11 (left) and CSSLdth8(SH527) (right). Plants were grown under natural LD. Photograph was taken when CSSLdth8(SH527) flowered. (B) Frequency distribution for flowering time in F2 population of CSSLdth8(SH527) and 93-11. Plants were grown under natural LD. Chi-square test checks the segregation ratio of flowering phenotypes in F2. (C) The red arrowhead denotes a 1-bp deletion in the SH527 DTH8 allele. F.S and STOP represent a frame-shift mutation and a premature stop codon in DTH8, respectively. Protein types are denoted as in a previous report (Wei et al., 2010). The black labels represent markers of fine mapping. The red label represents the location of SNP-1 and dCAPs8-2 markers in DTH8. (D) Expression of Ehd1 and Hd3a in 93-11 and CSSLdth8(SH527) as indicated by real-time qPCR results. Data are shown as means ± standard deviation; n = 3. Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 3 Flowering Phenotypes and Expression of Hd3a in Lines with or without Functional Hd1 and/or DTH8. (A) Flow diagram of NIL construction. (B) Flowering phenotypes of plants in the F2 generation of a cross between CSSLHd1(Nip) (DDHH) and CSSLdth8(SH527) (ddhh). Plants were grown under natural LD. Double asterisks above the graph indicate statistically significant differences (P < 0.01). (C) Expression of Hd3a in NILDH3-1, NILDh3-1, NILdH2-1, and NILdh1-1 at T0 (8:00 am) in LD and short-day conditions (SD). Data are shown as means ± standard deviation; n = 3. Double asterisks above the graph indicate statistically significant differences (P < 0.01). (D) Sequencing results of hd1DTH8, Hd1dth8 and hd1dth8 mutants in Dongjin background created using the CRISPR/Cas9 method. (E) Flowering phenotypes of hd1DTH8, Hd1dth8, hd1dth8, and Hd1DTH8. Plants were grown under LD and SD. Asterisks above the graph indicate statistically significant differences (*P < 0.05, **P < 0.01). (F) Expression of Hd3a in hd1DTH8, Hd1dth8, hd1dth8, and Hd1DTH8 at T0 (8:00 am) in LD. Data are shown as means ± standard deviation; n = 3. Double asterisks above the graph indicate statistically significant differences (P < 0.01). Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 4 Interaction between DTH8 Alleles and Hd1 Alleles in a Rice Variety Population in Natural LD. (A–D) Normal probability plots to test whether the data for different DTH8 and Hd1 allele combinations obey normal distribution. (E) Histogram based on empirical cumulative distribution and normal probability density function plotted using the data from varieties with Hd1DTH8 alleles and varieties with hd1DTH8 alleles. (F) Histogram based on empirical cumulative distribution and normal probability density function plotted using the data for varieties with Hd1dth8 alleles and varieties with hd1dth8 alleles. Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 5 Hd1 Can Interact with DTH8. (A) AD-Hd1 (Nipponbare) interacted with BD-DTH8 (93-11) in yeast two-hybrid assays. Empty vector expressing the AD domain alone was the negative control. (B and C) Interaction between Hd1-LUCn (Nipponbare) and DTH8-LUCc (93-11) under LD in LCI assays. Error bars represent standard deviation of six biological replicates. Double asterisks above the graph (C) indicate statistically significant differences (P < 0.01). (D) In vivo co-immunoprecipitation assays showing the association of Hd1 with DTH8 in N. benthamiana leaf cells. (E) BiFC assay for the interaction of DTH8 (93-11) and Hd1 (Nipponbare) in rice protoplasts. Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 6 Diurnal Expression Patterns of DTH8, Hd1, Ehd1, RFT1, and Hd3a. (A–P) Diurnal expression patterns of DTH8, Hd1, Ehd1, RFT1, and Hd3a in DJ, hd1, dth8, and hd1dth8 under LD and SD. Data are shown as means ± standard deviation; n = 3. (Q) Diurnal expression patterns of DTH8 in DJ under LD and SD. Data are shown as means ± standard deviation; n = 3. (R) Levels of DTH8 mRNA under various day-length conditions at T0 (8:00 am). Data are shown as means ± standard deviation; n = 3. Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 7 DTH8 Binding Is Enriched in the Hd3a Promoter and H3K27me3 Is Detected at Hd3a. (A) Scale diagram of the Hd3a promoter showing the positions of all CORE regions, CCAAT boxes, and the fragments in ChIP–qPCR assay. (B) Diagram of the wild-type and mutant (mut) fragments used for yeast one-hybrid assay. Substituted nucleotides in the mutant are shown in red. (C) Yeast one-hybrid assays to test the interaction between Hd1, DTH8, and the Hd3a promoter. (D) qPCR analyses of seven fragments in the Hd3a promoter in anti-FLAG-immunoprecipitated samples from 40-day-old 35S:6×FLAG-DTH8 leaves grown under LD. The ChIP values were normalized to their respective DNA inputs. Enrichment represents the value of (6×FLAG-DTH8 in fragments)/(6×FLAG-DTH8 in Actin1). Error bars represent standard deviation of three biological replicates. (E) qPCR analyses of seven fragments in the Hd3a promoter in anti-FLAG-immunoprecipitated samples from 40-day-old 35S:6×FLAG-DTH8 leaves grown under LD and SD. The ChIP values were normalized to their respective DNA inputs. Enrichment represents the value of (6×FLAG-DTH8 in fragments)/(6×FLAG-DTH8 in Actin1). Error bars represent standard deviation of three biological replicates. (F–I) qPCR analyses of seven fragments of Hd3a in anti-H3K27me3-immunoprecipitated and anti-H3-immunoprecipitated samples from 40-day-old leaves grown under LD and SD. Enrichment represents the value of (H3K27me3/H3 in fragments)/(H3K27me3/H3 in Actin1). Error bars represent standard deviation of three biological replicates. Asterisks above the graphs in (D) to (I) indicate statistically significant differences (*P < 0.05, **P < 0.01). Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions

Figure 8 Proposed Model for the Function of DTH8-Hd1 Complexes in Regulation of Hd3a Transcription. When DTH8 is absent due to loss-of-function mutation (dth8), Hd1 acts as an activator of Hd3a expression to promote flowering regardless of whether the plants are under LD or SD. In wild-type plants (WT) under SD, the DTH8-Hd1 module forms, but Hd1 continues to act as an activator of Hd3a expression to promote flowering. By contrast, in WT under LD, the DTH8-Hd1 module acts as a repressor of Hd3a expression. Red arrows indicate transcription activation. Gray bars indicate the transcription repression. White DTH8 protein indicates that an unknown mechanism may switch DTH8 activation in SD. Molecular Plant 2017 10, 948-961DOI: (10.1016/j.molp.2017.05.006) Copyright © 2017 The Author Terms and Conditions