DTH8 Suppresses Flowering in Rice, Inﬂuencing · PDF fileDTH8 Suppresses Flowering in Rice, Inﬂuencing Plant Height and Yield Potential Simultaneously1[W][OA] Xiangjin Wei, Junfeng

DTH8 Suppresses Flowering in Rice, Influencing PlantHeight and Yield Potential Simultaneously1[W][OA]

Xiangjin Wei, Junfeng Xu, Hongnian Guo, Ling Jiang, Saihua Chen, Chuanyuan Yu, Zhenling Zhou,Peisong Hu, Huqu Zhai, and Jianmin Wan*

State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Provincial Center of PlantGene Engineering, Nanjing Agricultural University, Nanjing 210095, China (X.W., J.X., H.G., L.J., S.C., C.Y.,Z.Z., J.W.); Chinese National Center for Rice Improvement, China National Rice Research Institute, Hangzhou310006, China (X.W., P.H.); and Institute of Crop Science, National Key Facility for Crop Gene Resources andGenetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China (H.Z., J.W.)

The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by manyquantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), wasfound to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of theCCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plantheight, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctionalDTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and itsprotein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulatethe transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T)under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (arice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, andthe natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these dataindicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor aswell as in the regulation of plant height and yield potential.

As food for more than half of the world’s popula-tion, rice (Oryza sativa) has always been a hot spot inplant science. Its yield is not only determined by spikenumber, grain weight, and number of grains perpanicle but also affected by plant height and floweringtime. Recent studies have shown much progress onexploring the mechanism for regulation of yield po-tential, plant height, and flowering time in rice. Somegenes regulating the rice yield trait have already beenidentified (Li et al., 2003; Ashikari et al., 2005; Fanet al., 2006; Song et al., 2007; Shomura et al., 2008;Wang et al., 2008; Weng et al., 2008). The control of rice

plant height is mostly related to the synthesis andregulation of the phytohormone, such as GA andbrassinolide (Ashikari et al., 1999; Yamamuroa et al.,2000; Honga et al., 2003; Sasaki et al., 2002, 2003; Itohet al., 2004; Tanabe et al., 2005). Photoperiod deter-mines flowering time as the most important envi-ronment signal in plants. Arabidopsis (Arabidopsisthaliana), the model for long-day (LD) species, hasbeen used to reveal how plants control floweringtime induced by photoperiod (Corbesier et al., 2007;Kobayashi and Weigel, 2008). A series of genes aboutphytochrome, clock, and flowering have been foundand a signal network has been built up. In LD condi-tions, CONSTANS (CO) gene in Arabidopsis, being atranscription factor and regulated by GIGANTEA (GI)gene, which is one of the circadian clock genes (Parket al., 1999; Huq et al., 2000; Sothern et al., 2002),activates the expression of FLOWERING LOCUS T (FT;Kardailsky et al., 1999; Kobayashi et al., 1999). Afterthe FT protein moves to the shoot apex, it binds to FD,which is another transcription factor, to trigger genesthat determine floral organ identity and then induceflowering (Abe et al., 2005; Wigge et al., 2005; Corbesieret al., 2007). Meanwhile, the short-day (SD) plant ricehas a similar photoperiodic control of flowering path-way. Signals from light and circadian clocks are sent toOsGI (a rice ortholog of Arabidopsis GI) and OsGIregulates the expression of Heading date1 (Hd1, a rice

1 This work was supported by grants from the 863 Program ofChina (grant nos. 2006AA100101, 2006AA10Z1A5, 2006BAD13B01,and 2009AA101101), the National Natural Science Foundation ofChina (grant nos. 30871497 and 30571142), Jiangsu Cultivar Devel-opment Program (grant no. BE2009301–3), Doctorial Foundation ofEducation Department (grant no. 20090097110011), and the 111Project (grant no. B08025).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Jianmin Wan ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.110.156943

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ortholog of Arabidopsis CO) and OsMADS51 (Yanoet al., 2000; Hayama et al., 2003; Kim et al., 2007). Whenrice is in SD conditions,Hd1 activates flowering by up-regulating Hd3a (a rice ortholog of Arabidopsis FT;Kojima et al., 2002). And OsMADS51 activates Hd3athrough Early heading date1 (Ehd1), in which Ehd1encodes a B-type response regulator and has no obvi-ous counterpart in Arabidopsis (Doi et al., 2004; Kimet al., 2007). By contrast, Hd1 represses flowering bydown-regulating Hd3a expression under LD condi-tions (Kojima et al., 2002). Additionally, RID1/OsID1/Ehd2, which is homologous to maize (Zea mays) Inde-terminate1 (ID1), promotes flowering in both SD andLD conditions by up-regulating Ehd1 (Matsubara et al.,2008; Park et al., 2008; Wu et al., 2008). Ghd7, encodinga CCT domain protein, has been proven to have majoreffects on an array of traits in rice, including number ofgrains per panicle, plant height, and flowering time(Xue et al., 2008); it represses Ehd1 and Hd3a expres-sion and then delays flowering in LD conditions.

As one of the most common promoter elementsfound in a large number of genes in animals, fungi,and plants, the CCAAT box is recognized by the HAPcomplex, which consists of three subunits: HAP2(NF-YA/CBF-B), HAP3 (NF-YB/CBF-A), and HAP5(NF-YC/CBF-C; Maity and de Crombrugghe, 1998;Mantovani, 1999). In yeast (Saccharomyces cerevisiae)and mammals, each of the HAP subunits is encodedby a single gene (Mantovani, 1999), different fromthose in plants, in which all subunits are encoded bygene families (Gusmaroli et al., 2001, 2002). TheArabidopsis genome encodes 10 AtNF-YA, 13 AtNF-YB, and 13 AtNF-YC subunits (Gusmaroli et al., 2002).The rice genome encodes 10 OsHAP2, 11 OsHAP3,and seven OsHAP5 subunits (Thirumurugan et al.,2008). Each subunit of the HAP complex contains anevolutionary conserved domain that mediates DNAbinding or protein-protein interaction. Recent researchsuggests that the functional domain conserved withinHAP2 and the CCT domain of CO share importantresidues, which are critical to the function of the CCTdomain. CO might replace AtHAP2 in the HAP com-plex to form a trimetric CO/AtHAP3/AtHAP5 com-plex and flowering was delayed by overexpression ofAtHAP2 or AtHAP3 in Arabidopsis (Wenkel et al.,2006). Also, two NF-YB/ HAP3 genes, LEAFY COTY-LEDON1 and LEC1-LIKE, were shown to be importantregulators of embryogenesis (Lotan et al., 1998; Kwonget al., 2003). In rice, OsHAP3A or its homologs,OsHAP3B and OsHAP3C, were shown to control chlo-roplast biogenesis (Miyoshi et al., 2003). However, thefunctions of other members of the NF-YB/HAP3 genefamilies and all members of theNF-YA/HAP2 andNF-YC/HAP5 in Arabidopsis and rice are still unknown.

In this study, we identified a rice HAP3 gene, DTH8,by map-based cloning. Our data showed that DTH8suppressed rice flowering under LD conditions andwe surmise that it probably plays an important role inthe regulation of plant height and yield potential inrice.

RESULTS

A Chromosome Segment of DTH8 Has a RemarkableEffect on Flowering Time, Plant Height, and Yield

Quantitative trait loci (QTL) analysis for days toheading was conducted over 5 years in natural LD(NLD) conditions (daylength . 14 h) based on arecombinant inbred line population (RIL), derived fromtwo parents, cvAsominori (japonica) and cv IR24 (indica;Supplemental Fig. S1). One major QTL, DTH8, nearmarker R2976 on the short arm of chromosome 8 hasbeen detected in all 5 years (Supplemental Table S1).Several chromosome segment substitution lines(CSSLs), such as CSSL8 and CSSL61, carrying a chro-mosome segment of IR24 on DTH8 under the Asomi-nori genetic background, showed earlier heading andits plant height, number of grains per panicle, yield,and dry weight per plant decreased significantly inNLD condition compared with Asominori (Fig. 1;Table I; Supplemental Fig. S2; Supplemental TableS2), but no significant differences in these agronomictraits have been found in natural SD (NSD) conditions(daylength about 11.6 h; Fig. 1; Supplemental TableS3). There was no difference in tiller number betweenCSSL61 and Asominori under either NLD or NSDconditions (Fig. 1; Supplemental Tables S2 and S3).

DTH8 Encodes a Putative HAP3 Subunit of theCCAAT-Box-Binding Transcription Factor

An analysis of two secondary F2 populations, de-rived from the cross between CSSL8, CSSL61, andAsominori, showed that earlier heading and laterheading plants of the two secondary F2 populationssegregated as 62:164 and 56:150, respectively (x2 = 0.71,0.52 , x2

2,0.05 = 3.84, P . 0.05; Supplemental Fig. S3),which suggests thatDTH8 is a single Mendelian factor.Then, map-based cloning of DTH8 was performed. Ahigh-resolution linkage analysis demonstrated thatDTH8 is delimited within the interval between twoinsertion-deletion (Ind) markers, Ind8-47 and Ind8-15.This region was about 47 kb in the bacterial artificialchromosome contig OSJNBa0054L03 (http://www.gramene.org; Fig. 2A).

The genome annotation software, RiceGAAS (http://ricegaas.dna.affrc.go.jp), predicts 12 open reading frames(ORFs) in this region (Fig. 2A). One of them, ORF4,encodes a putative HAP3/ NF-YB/CBF-A subunit of theCCAAT-box-binding transcription factor, and has beenannotated as a flowering-time gene, Hd5, in the database(Fig. 2C). There are 11 homologous genes (OsHAP3A–OsHAP3K) encodingHAP3 subunits in the genomeof riceandORF4 isnamedOsHAP3H (Thirumuruganet al., 2008;Fig. 2D; Supplemental Fig. S4). Without any intron, thereading frame of OsHAP3H totals 894 bp, encoding aprotein of 297 amino acids in cv Asominori (Fig. 2, B andC). However, in cv IR24, 1-bp deletion was found in aposition 322 bp away from start codon, causing a frame-shift andpremature terminationof translationand leadingto a mutant protein with only 125 amino acids (Fig. 2B).

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To confirm that OsHAP3H is responsible for thephenotype of DTH8, the OsHAP3H cDNA from cvAsominori was expressed under CSSL61 background.The cDNAwas inserted into pPZP2H-lac binary vector(Fuse et al., 2001) and 13 independent transgenic T0lines were generated, which were planted in NSDconditions. The segregation ratio between transgene-positive and transgene-negative plants in T1 familiesfrom T0 plants with single-copy transgene was about3:1 (Table I). Perfect cosegregation between the trans-gene and the phenotype was also observed (Fig. 3;Table I): comparing with transgene-negative plants,days to heading, plant height, and number of grainsper panicle of all transgene-positive plants increasedsignificantly. Taken together, DTH8, which encodes aputative HAP3/NF-YB/CBF-A subunit of the CCAAT-box-binding transcription factor, has a large effect onflowering time, plant height, and yield potential in rice.

Expression Pattern and Subcellular Localization of DTH8

Quantitative real-time PCR (QRT-PCR) analyseswere done with total RNA isolated from leaves, leafsheaths, culms, young panicles, and roots from cvAsominori grown under NLD conditions (Fig. 4A).

The results revealed that DTH8 was expressed in allexamined tissues: The expressions were higher inroots, young panicles, and leaves than those in leafsheaths and culms. To reconfirm the results above, theGUS gene driven by the DTH8 promoter was trans-formed into rice (Fig. 4B). Staining of transgenic plantsverified again theDTH8 expression in the same tissuesexamined with QRT-PCR. The staining also showedmore accumulation in roots, young panicles, andleaves, which matched the QRT-PCR findings.

Subcellular localization of DTH8 protein was car-ried out by fusing DTH8 and GFP, which was drivenby the cauliflower mosaic virus 35S promoter (35S::DTH8:GFP) and delivered into onion (Allium cepa)epidermal cells by particle bombardment. The GFPsignal was localized in the nucleus (Fig. 4C), indicatingthat DTH8 is a nuclear protein. This result also sup-ports the hypothesis that DTH8 acts as a subunit of theCCAAT-box-binding transcription factor.

DTH8 Suppresses Flowering by Down-Regulating Ehd1and Hd3a

To identify potential downstream genes that areregulated by DTH8, QRT-PCR was performed to

Figure 1. Phenotypes of Asominori andCSSL61. A, Photos of Asominori andCSSL61, taken when CSSL61 reachedmaturity. B, Photos of Asominori andCSSL61, taken when Asominori reachedmaturity. C, Main panicles of Asominoriand CSSL61. D, Grains from the mainpanicles of Asominori and CSSL61. A toD, Asominori and CSSL61 were grownunder NLD conditions. E, Photos ofAsominori and CSSL61, taken underNSD conditions. A to E, The left isAsominori and right is CSSL61. F, Daysto heading of Asominori and CSSL61 indifferent daylength conditions. G, Thegenotype of CSSL61.

DTH8 Suppresses Flowering in Rice

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analyze the expression levels of DTH8 and sevenflowering-related genes (Hd1, Ehd1, Hd3a, Ghd7,OsGI, OsMADS51, and RID1) in both Asominori andCSSL61. Leaf samples from 40-d-old plants grownunder SD, LD, and NLD conditions were checked. Nodifferences in DTH8 expression were found betweenAsominori and CSSL61 under all three conditions (Fig.5; Supplemental Fig. S5), suggesting that the naturalmutation in the coding region does not significantlyaffect the expression of DTH8, and neither did thedifferences in OsGI, RID1, OsMADS51, and Ghd7 ex-pressions between Asominori and CSSL61 in all con-ditions (Fig. 5; Supplemental Fig. S5). For Hd1, thenatural mutation of DTH8 slightly induced its downexpression in SD conditions but not under LD andNLD (Fig. 5; Supplemental Fig. S5). For Ehd1 andHd3agenes, this mutation led to a great reduction of tran-scription in CSSL61 under LD and NLD (Fig. 5; Sup-plemental Fig. S5). These results suggest that Ehd1 andHd3a possibly function downstream of DTH8, whichsuppresses rice flowering in LD condition by down-regulating Ehd1 and Hd3a. However, the expression ofEhd1was only slightly lower, whereasHd3a expressionwas not changed at all in SD conditions (Fig. 5;Supplemental Fig. S5). This result was consistent withthe observation that flowering time was not signifi-cantly different between Asominori and CSSL61 in SDconditions (Fig. 1; Supplemental Table S3).

Also, the diurnal expression patterns of DTH8,OsGI, Hd1, Ehd1, Hd3a, and Ghd7 were checked inboth Asominori and CSSL61 (Supplemental Fig. S6).The diurnal pattern of DTH8 is similar to that of OsGIin both LD and SD conditions. As in the single mea-surements at the onset of the light phase, severereductions in Ehd1 and Hd3a mRNAwere detected inboth day and night cycles under LD conditions inAsominori. Meanwhile, little change was found in thediurnal patterns of OsGI, Hd1, Ehd1, Hd3a, and Ghd7expressions between Asominori and CSSL61 in bothLD and SD conditions.

The Expression of DTH8 Is Not Influenced by Hd1and Ghd7

Previous studies show that the expression of Ehd1andHd3a could be down-regulated byGhd7 (Xue et al.,2008) and Hd3a can also be suppressed by Hd1 underLD conditions (Kojima et al., 2002; Hayama et al.,2003). In this study, our data showed that under LDconditions, DTH8 could negatively influence the ex-pression of Ehd1 and Hd3a but not Hd1 and Ghd7 (Fig.5; Supplemental Fig. S5). To examine whether theexpression of DTH8 is influenced by Hd1 and Ghd7,DTH8 expression was examined in Hd1 and Ghd7near-isogenic lines (NILs) grown for 40 d under LDconditions. As shown in Figure 6A and SupplementalFigure S7A, the expression levels of DTH8 and Ghd7,compared with those in Nipponbare, were not changedin NIL (hd1) grown under LD conditions. However,Ehd1 and Hd3a were suppressed by Hd1 in Nippon-bare. Similarly, no changes were found in the levels ofDTH8 and Hd1 mRNA in the Ghd7 NILs under LDconditions. But the expressions of Ehd1 and Hd3awerealso suppressed by Ghd7 (Fig. 6B; Supplemental Fig.S7B). These results indicate that DTH8 suppresses riceflowering by down-regulating the expression of Ehd1and Hd3a, and that the expression of DTH8, Hd1, orGhd7 is independent of each other under LD condi-tions.

DTH8 Regulates the Stem Growth

During maturity, the average height of CSSL61 andtransgene-negative plants was approximately 84% ofAsominori and transgene-positive plants (Figs. 1Band 3B; Table I). The internode elongation patternsbetween CSSL61 and Asominori were compared (Fig.7; Supplemental Fig. S8) and the data showed that thepanicles and internodes of CSSL61 were remarkablyshorter than those of Asominori. Meanwhile, thediameter of stems between them was almost thesame. To determine whether the dwarf phenotype

Table I. Phenotypes of Asominori, CSSL61, transgene-positive, and transgene-negative segregants inthe T1 generation derived from T0 plants with single-copy transgenes under NLD conditions

Family 1 and 2 are the T1 plants derived from two independent T0 plants with single-copytransgene. Data are mean 6 SE. (+) and (2) indicate transgene-positive and transgene-negative, re-spectively.

GenotypeNo. of

Plants

No. of Days

to Heading

Plant Height

in cm

No. of Spikelets

per Panicle

Asominori 20 93.0 6 1.3 85.3 6 3.2 88.7 6 10.9CSSL61 20 80.0 6 2.1 71.3 6 3.0 59.2 6 5.9P value (t test) 2.21 3 10214 1.80 3 1028 3.21 3 1025

Family 1 (+) 31 89.8 6 2.8 81.6 6 6.3 90.9 6 19.3Family 1 (2) 12 80.5 6 3.4 69.2 6 7.4 63.1 6 12.2P value (t test) 1.17 3 1027 3.65 3 1025 1.63 3 1026

Family 2 (+) 33 92.4 6 1.4 78.4 6 4.2 99.7 6 11.6Family 2 (2) 13 80.0 6 2.6 65.6 6 7.4 56.6 6 13.3P value (t test) 5.08 3 10211 1.43 3 1025 1.29 3 1029

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of the CSSL61 results from defective cell divisionand/or cell elongation, longitudinal sections of inter-nodes III and IV of CSSL61 culms were comparedwith their Asominori counterparts. As shown inFigure 7, C, F, and G, the total cell number in the yaxes in internodes III and IV of CSSL61 was not

significantly less than that of Asominori, while thecell length of CSSL61 was significantly shorter. Thisresult suggests that internode cell elongation is themain reason for the dwarf phenotype of CSSL61 andthat DTH8 probably plays an important role in stemgrowth and development.

Figure 2. Map-based cloning of DTH8. A, Fine mapping of DTH8. B, Structure of DTH8. Vertical lines without labels representsingle-base substitutions between Asominori and IR24. Small rectangular boxes and arrowheads represent deletions andinsertions, respectively. C,DTH8 cDNA and predicted amino acid sequence. Letters with an asterisk indicate 1-bp deletion IR24,and the amino acids with gray background represent the conserved domains for DNA binding or protein-protein interaction. F.S.and STOP represent frameshift mutation and create premature stop codon, respectively. D, A phylogenic tree of HAP3 proteinfamilies in rice, Arabidopsis, yeast (ScHAP3, NP_009532), and human (HsNF-B, L06145). The unrooted tree was generatedbased on the amino acid sequences of the conserved domain using the program DNAMAN. The scale represents the substitutionpercentage per site, and the similarity to DTH8 in the conserved domain of each HAP protein is indicated in parentheses.





Rice Varieties with Nonfunctional DTH8 Have WeakerSensitivity to Photoperiod

We investigated the diversity of the coding se-quences of DTH8 among 40 rice varieties from awide geographic range in Asia (Supplemental TableS4). The DTH8 alleles in these varieties were groupedinto nine types and eight distinct proteins were iden-tified (Fig. 8). Frameshift mutations or premature stopscaused by the deletions of some nucleotides werefound in types 7, 8 (IR24), and 9 (Fig. 8). So, the DTH8proteins of these three allele types seem nonfunctional.For other types, the protein of type 2 is same as that of

type 1 (Asominori), and types 3 to 6 just have severalSNPs and indels that could cause single amino acidsubstitution, addition, or deletion (Fig. 8). Therefore,the proteins encoded by types 2 to 6 are probablyfunctional. To confirm this, the DTH8 alleles of type 4(from cv Kasalath), type 5 (from cv 93-11), and type 6(from cv IR64) were transformed into the CSSL61 (withnonfunctional DTH8 allele from IR24) background.The results showed that all transgene-positive plantsin the T1 families were tall and late heading with largepanicles, whereas all transgene-negative plants havephenotypes with opposite features (Supplemental Fig.S9; Supplemental Table S5). We can thus separate thecultivars into two groups, functional and nonfunc-tional DTH8 alleles (Fig. 8; Supplemental Table S4).The photoperiod sensitivity (PS) of flowering time inthese varieties was also analyzed using PS index (PSindex = [DTHNLD 2 DTH

SD]/DTHNLD; DTH, NLD, and

SD represent days to heading, NLD, and SD condi-tions, respectively; Supplemental Table S4). The resultsshowed that cultivars that carry the functional DTH8alleles tended to have larger PS indexes and that theirflowering times were more sensitive to photoperiod,whereas those carrying nonfunctional DTH8 allelestended to show smaller PS indexes and less photope-riod-sensitive flowering times (Fig. 8; SupplementalTable S4).

DISCUSSION

DTH8 Has Pleiotropic Effects

Yield, plant height, and flowering time are the threemost important agronomic traits in rice. Grain yield ismainly determined by factors such as spike/tiller

Figure 3. Comparison of phenotype of the DTH8 transformants andCSSL61 plants. A, Transgene-positive T1 plant (left) and CSSL61 plant(right). B, Transgene-positive T1 plant (left) and transgene-negative T1plant (right). C, Main panicles of transgene-positive T1 plant (left) andtransgene-negative T1 plant (right).

Figure 4. Expression pattern ofDTH8 and subcellular localizationof its protein. A, Tissue-specific ex-pression pattern revealed by QRT-PCR. RNAwas isolated from leaves(L), leaf sheaths (S), culms (C),young panicles (P), and roots (R)from Asominori when it was head-ing in NLD conditions. B, GUS ex-pression in different tissues drivenbyDTH8 promoter under NLD con-ditions. a to f, Root, culm, node, leafblade, leaf sheath, and young pan-icles. C, Nuclear localization ofDTH8. a and e, Bright-field imagesof onion epidermal cells. b, Nuclearlocalization of DTH8-GFP fluores-cence. c, The same cells stainedwith DAPI. d, The merged image ofa, b, and c. f, Onion epidermal cellsbombardedwith the construct havingGFP alone as the control. g, Themerged image of e and f.

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number, number of grains per panicle, and grainweight. Recently, some genes related to yield havebeen cloned, e.g. MOC1, which initiates axillary budsand plays an important role in the control of ricetillering (Li et al., 2003); Gn1a controls the number ofgrains per panicle (Ashikari et al., 2005); GS3, GW2,and qSW5/GW5 regulate grain size (grain length andgrain width) and grain weight (Fan et al., 2006; Songet al., 2007; Shomura et al., 2008; Weng et al., 2008);and GIF1 controls rice grain filling (Wang et al., 2008).More genes for plant height in rice have been iden-tified (Ashikari et al., 1999; Yamamuroa et al., 2000;Sasaki et al., 2002, 2003; Honga et al., 2003; Itoh et al.,2004; Tanabe et al., 2005). The molecular geneticpathway for flowering time has been under elucida-tion in rice since the isolation of many correlativegenes or QTLs (Yano et al., 2000; Kojima et al., 2002;Doi et al., 2004; Kim et al., 2007; Wu et al., 2008).Although there are close correlations among yield,plant height, and flowering time, most of these genes

were reported to be regulating only one of the threetraits. Recently, one QTL, Ghd7, has been proven tohave large pleiotropic effects on an array of traits,including grain number, flowering time, and plantheight (Xue et al., 2008). In this study, a rice HAP3gene, DTH8, was isolated by map-based cloning. Itslarge pleiotropic effects were likewise demonstrated.DTH8 also delayed rice flowering by negativelyinfluencing the expression of Ehd1 and Hd3a, beingindependent of Hd1 under LD conditions. Simulta-neously, it plays an important role in regulating plantheight and grain number. Meanwhile, the differencesbetween DTH8 and Ghd7 were also very obvious.Tiller number in Ghd7 NILs with functional or non-functional alleles differed from each other (Xue et al.,2008); those between Asominori and CSSL61 are thesame (Fig. 1; Supplemental Tables S2 and S3). Ghd7regulates the height through total cell number in the yaxes of internodes and it also influences culm thick-ness, whereas DTH8 regulates height by altering cell

Figure 5. The expression of DTH8and other flowering-time genes inAsominori and CSSL61. QRT-PCRwas performed with total RNA fromleaves of 40-d-old plants under SDand LD conditions. Samples werecollected at the initiation of the lightphase (ZT 0 h). These experimentswere repeated at least three times.

Figure 6. The expression of DTH8and other flowering-time genes in theNILs of Hd1 and Ghd7 under LDconditions. A, The expression in cvNipponbare (Nip) and NIL (hd1;carries hd1 introgressed from cvKasalath in a cv Nipponbare geneticbackground). B, The expression inNIL (Ghd7; carries functional Ghd7fromMinghui63) and NIL (ghd7; car-ries nonfunctional ghd7 from Zhen-shan97). QRT-PCR was performedwith total RNA from leaves of 40-d-old plants under LD conditions. Sam-ples were collected at the initiation ofthe light phase (ZT 0 h). These exper-iments were repeated at least threetimes.





elongation in the internodes, but keeps culm thick-ness stable (Fig. 7).

DTH8 Is a Novel Flowering Suppressor

Rice is a SD plant and most rice cultivars could beinduced to flower more rapidly under SD conditionsthan under LD conditions. In SD conditions, signalsfrom light and circadian clocks are received by OsGI,and it regulates the expression of Hd1 and OsMADS51(Izawa et al., 2003; Kim et al., 2007). Hd1 induces riceflowering by up-regulating Hd3a expression (Kojimaet al., 2002), while OsMADS51 promotes flowering byactivating the expression of Hd3a through Ehd1 (Doiet al., 2004; Kim et al., 2007). In LD conditions, theexpression of Hd3a is suppressed by Hd1 (Hayamaet al., 2003), and Ehd1 andHd3a are also suppressed byGhd7 (Xue et al., 2008), which accounts for the lateflowering in rice. Additionally, RID1/OsID1/Ehd2promotes flowering under both SD and LD conditionsby up-regulating Ehd1 (Matsubara et al., 2008; Parket al., 2008; Wu et al., 2008). In this study, DTH8 wasfound to delay rice flowering time by negativelyinfluencing the expression of Ehd1 and Hd3a in LDconditions (Fig. 5; Supplemental Fig. S5). But, underSD conditions, the expression of Hd3a and rice flower-ing time are not influenced by DTH8, and neither arethe expressions of RID1, OsGI, Hd1, and Ghd7 in LDconditions (Fig. 5; Supplemental Fig. S5). Meanwhile,the expression of DTH8 is independent of Hd1 andGhd7 in LD conditions (Fig. 6; Supplemental Fig. S7),suggesting that DTH8 is a flowering suppressor bydown-regulating the expression of Ehd1 and Hd3aunder LD conditions. There are many studies to havereported that Ehd1 locates in the upstream ofHd3a and

could promote its expression (Doi et al., 2004; Kimet al., 2007; Matsubara et al., 2008; Park et al., 2008; Wuet al., 2008). At the same time, some other reports showthat the flowering suppressor could repress Hd3aexpression by down-regulating Ehd1 and then inhibitsflowering (Xue et al., 2008). So we propose that DTH8is upstream of Ehd1, which in turn is upstream ofHd3ain the pathway (Fig. 9). The diurnal expression patternof DTH8 is similar to that of OsGI in both LD and SDconditions (Supplemental Fig. S6), suggesting thatDTH8 probably locates downstream of OsGI in thepathway that regulates photoperiodic flowering time(Fig. 9). Because there is no Ehd1 ortholog in Arabi-dopsis (Doi et al., 2004), the DTH8-Ehd1-Hd3a relationprobably represents a distinct pathway that does notexist in Arabidopsis.

Previous research shows that Hd1 and Ehd1 regulatethe expression of Hd3a through two different path-ways (Doi et al., 2004). However, the results from thisstudy showed that the expression of Ehd1 is sup-pressed by Hd1 in LD conditions (Fig. 6A; Supple-mental Fig. S7A). So Ehd1 may be the integrator ofdifferent pathways in the regulation of flowering,which is down-regulated by Hd1, Ghd7, and DTH8but up-regulated by RID1. Then, the expression ofHd3a or its orthologs was activated by Ehd1 and thetransition from vegetative development to floral de-velopment in rice was induced under LD conditions(Fig. 9).

Possible Reason for the Pleiotropic Effects of DTH8

The protein encoded by DTH8 is a putative HAP3/NF-YB/CBF-A subunit of the HAP complex, Os-HAP3H (Fig. 2), which binds to CCAAT box that is

Figure 7. The differences of theculms and scanning electron mi-croscopic (SEM) observation of in-ternodes between Asominori andCSSL61 plants. A, Main culms ofAsominori (left) and CSSL61 (right)plants. Arrows indicate the posi-tions of nodes. B, The differencesof panicles and internodes of mainculms between Asominori (left) andCSSL61 (right) plants. P, Panicle.Those from I to V indicate thecorresponding internodes from topto bottom. C, Total cell number ofinternodes III and IV in y axis. Dand E, SEM of transverse sections ofthe middle part of internode IV ofAsominori (D) and CSSL61 (E)plants at the mature stage. F andG, SEM of longitudinal sections ofthe middle part of internode IV ofAsominori (F) and CSSL61 (G)plants at the mature stage.

Wei et al.




the cis-acting element in about 25% of eukaryotic pro-moters (Maity and de Crombrugghe, 1998; Mantovani,1999). The HAP complex consists of three subunits:HAP2/NF-YA/CBF-B, HAP3/NF-YB/CBF-A, andHAP5/NF-YC/CBF-C. Each subunit of the HAP com-plex contains a conserved domain that is responsiblefor DNA binding or protein-protein interaction. Al-though many genes encoding HAP subunits havebeen found so far (Edwards et al., 1998; Gusmaroliet al., 2001, 2002; Thirumurugan et al., 2008), only afew of them have been studied (Lotan et al., 1998;Meinke, 1992; West et al., 1994; Kwong et al., 2003;Miyoshi et al., 2003). In this study, a rice HAP3 gene,DTH8, has been proven to play an important role inthe regulation of flowering time, plant height, andyield potential. Because of the existence of multipleHAP2 and HAP5 subunit orthologs, the HAP com-plexes OsHAP2/DTH8/OsHAP5 should not be theonly one that may bind to the CCAAT boxes in thosegenes working in different metabolic pathways. Thegenes controlling the number of primary branches andsecond branches are regulated by the OsHAP2/DTH8/OsHAP5 complexes, which could influencethe number of grains per panicle. Rice height wasaffected by DTH8 or OsHAP complexes mainlythrough the regulation of internode cell elongation(Fig. 7). According to previous reports, the control ofrice height is mostly related to the synthesis andregulation of phytohormones (Ashikari et al., 1999;Yamamuroa et al., 2000; Sasaki et al., 2002, 2003; Hongaet al., 2003; Itoh et al., 2004; Tanabe et al., 2005). TheOsHAP2/DTH8/OsHAP5 complexes may be in-volved in the biosynthesis or degradation of phyto-hormones.In this study, we found that DTH8 delayed rice

flowering time by down-regulating the expression ofEhd1 and Hd3a in LD conditions (Fig. 5). But theexpressions of DTH8, Hd1, and Ghd7 were not influ-enced by each other under LD conditions (Figs. 5 and

6), suggesting that the down-regulation of Ehd1 andHd3a by DTH8 may be independent of Hd1 and Ghd7under these conditions (Fig. 9). Recent research hasshown that the conserved domain of HAP2 and theCCT domain of CO are similar to each other, wherethe most conserved residues are very important for thefunctioning of the CCT domain. CO could replaceAtHAP2 in the HAP complex to form a trimetriccomplex, CO/AtHAP3/AtHAP5 (Wenkel et al., 2006).In LD conditions, flowering was postponed by over-expression of AtHAP2 or AtHAP3 in Arabidopsis.Meanwhile, FT expression was going down while thelevel of CO remained stable. The mRNA abundance ofAtHAP3a shows a diurnal rhythm and is regulated bythe flowering-time gene GI (Wenkel et al., 2006). Therice flowering genes Hd1 and Ghd7 also have the CCTdomains (Yano et al., 2000; Xue et al., 2008) so theymay

Figure 8. Different types of DTH8 and their relationships with PS of flowering time. Type 1 is the genotype of cv Asominori.Polymorphic nucleotides in other cultivars are indicated by different colors. Deletion and insertion sites are indicated by whiteand black arrowheads, respectively. The number of cultivars and the mean value of PS index with each type of sequence (typesfrom 1–9) are shown in the column at the right, with the numbers for loss-of-function types in red. The amino acid sequences oftypes 1 and 2 are identical. F.S. is frameshift and STOP is premature stop.

Figure 9. A proposed model for the initiation and integration of theflowering pathways in rice under LD conditions.





be able to replace OsHAP2 in the HAP complex. Thus,we speculate that the down-regulation of Ehd1 andHd3a by DTH8, Hd1, and Ghd7 might be mediated byforming the possible complexes Hd1/DTH8/OsHAP5and Ghd7/DTH8/OsHAP5. Of course, more evi-dences are needed to test this hypothesis.

Relationship between DTH8 and PS

A high degree of polymorphism in the DTH8 se-quences was identified, some of which cause frame-shift mutations or create premature stop codons. TheDTH8 alleles in the core collection were grouped intonine types and eight distinct proteins were identified(Fig. 8). All the cultivars were separated into groupswith functional or nonfunctional DTH8 alleles, de-pending on the results of transgenic analyses (Fig. 8;Supplemental Fig. S9; Supplemental Tables S4 and S5).Being a SD plant, the flowering time of rice is sensitiveto variation in photoperiod induced by SD conditionsand blocked by LD conditions. But with the longperiod of introduction and domestication, some ricevarieties have become insensitive to photoperiod var-iation and flowering time of rice show little or even nodifference when they are grown under either SD or LDconditions. Varieties with weak PS are usually grownin high-latitude area or in mid- and low-latitude areaas early season rice.

The PS of flowering time for 40 varieties was alsoanalyzed by looking at the PS index (SupplementalTable S4). The results showed that flowering times ofcultivars that carry functional DTH8 alleles are moresensitive to photoperiod, whereas those with nonfunc-tional DTH8 alleles are less sensitive (Fig. 8; Supple-mental Table S4). However, there are still someexceptions, such as cv Jingnong1, cv XX-4, and cv 93-11. The flowering times of these varieties are weaklysensitive to photoperiod, even though they carry thefunctional DTH8 alleles (Supplemental Table S5). Thepossible reason is that these cultivars may containother unknown genes that can weaken their PS. Thecultivars having nonfunctional DTH8 alleles andshowing weaker PS are grown as early season rice inmid- and low-latitude areas (Supplemental Table S5).Thus, it might be a better choice to breed rice cultivarswith nonfunctionalDTH8 alleles in high-latitude areasor as early season rice in mid- and low-latitude areas.The cultivars with weaker PS would be able to adaptwell to LD conditions. Similarly, while breeding singleor late-season rice for the mid- or low-latitude areas,cultivars with functional DTH8 alleles should be cho-sen because the flowering of these cultivars could beinduced by the SD conditions and their grain yieldcould be increased by DTH8.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

A RIL and several CSSLs derived from two parents, cv Asominori

(japonica) and cv IR24 (indica; Kubo et al., 1999; Supplemental Fig. S1), were

grown in Nanjing NLD conditions (daylength . 14 h). The materials that

include Asominori, CSSL61, Nipponbare, NIL (hd1), NIL (Ghd7), and NIL

(ghd7) for the expression of DTH8 and other flowering genes were grown in

NLD, LD (15 h light/9 h dark), and SD (9 h light/15 h dark) conditions.

Asominori and CSSL61, for the analysis of diurnal expression patterns of

flowering genes, were first grown in NLD conditions for 30 d and then

transferred to LD or SD conditions for 10 d. The transgene-positive T0 and T1

plants were grown in NSD (about 11.6 h light/12.4 h dark) conditions (Hainan

Province, China) and NLD conditions, respectively. The 40 varieties from Asia

for genotyping the DTH8 locus were grown under NLD and SD conditions.

Mapping for DTH8

The RILs and several CSSLs derived from Asominori (japonica) and IR24

(indica; Tsunematsu et al., 1996; Kubo et al., 1999) were used for primary

mapping of the DTH8 locus (Supplemental Fig. S1). The secondary F2

population containing 15,000 plants was constructed from the cross between

CSSL61 and Asominori and 2,000 earliest plants were selected to fine map the

DTH8 locus, using the approach described by Zhang et al. (1994). DTH8 was

first localized to a 2.3-cM interval between simple sequence repeat (SSR)

markers RM22475 and RM5556, based on 200 earliest plants. After more than

nine SSR or InDel markers were developed between RM22475 and RM5556, 40

recombinant plants were selected andDTH8was finally narrowed to the 47-kb

region between Ind8-47 and Ind8-15 (Fig. 2A). The molecular markers

including SSR and InDel markers used for fine mapping DTH8 are shown

in Supplemental Table S6.

Vector Construction and Transformation

The full-length coding region of DTH8 was isolated by PCR with primers

8 to 16 (Supplemental Table S7) from Asominori, Kasalath, 93-11, and IR64,

then subcloned into the pPZP2H-lac binary vector (Fuse et al., 2001). The

resultant plasmid was introduced into CSSL61 by means of Agrobacterium-

mediated transformation (Hiei et al., 1994). The genotype of transgenic plants

was determined by PCR amplification of the hygromycin phosphotransferase

gene (hpt) and the analysis of hygromycin resistance.

A 2.5-kb sequence of DTH8 promoter region was isolated by PCR ampli-

fication with primers Pro-14 (Supplemental Table S7) from Asominori and

then subcloned into the pCAMBIA1381Z binary vector. The resulted plasmid

was transformed into rice (Oryza sativa) through Agrobacterium-mediated

transformation, and the transgenic plants were analyzed by GUS staining

assay as described (Scarpella et al., 2003).

RNA Extraction and QRT-PCR

Total RNA from leaves, leaf sheaths, culms, roots, and young panicles were

isolated using an RNA extraction kit following the manufacturer’s instruction

(Beijing Dingguo Biotechnology Co. Ltd., http://www.dingguo.com). First-

strand cDNA was reverse transcribed from DNase I-treated RNA with oligo

(dT) as the primer. Gene expression was measured by QRT-PCR and semi-

quantitative RT-PCR using Ubiquitin and Actin gene as internal controls. The

QRT-PCR and semiquantitative PCR primers for Actin, DTH8, OsGI, Hd1,

Hd3a, Ehd1, Ghd7, RID1, and OsMADS51 are listed in Supplemental Table S8.

The QRT-PCRwas carried out in a total volume of 25 mL containing 2 mL of the

reverse-transcribed product above, 0.2 mM of each primer and 13 SYBR green

PCR master mix (TaKaRa Co. Ltd., http://www.takara.com.cn). The PCR was

performed with a Bio-Rad iCycler (http://www.bio-rad.com/) using the

following program: 95�C for 30 s, then 40 cycles of 95�C for 5 s, 60�C for 34 s.

Changes in gene expression were calculated via the DDCT method. The

semiquantitative PCR conditions were 95�C for 3 min, then 26 cycles (cycle

numbers for Actin were 26; DTH8 and OsGI were 28; others were 30–35) of

95�C for 30 s, 58�C for 30 s, and 72�C for 30 s.

Subcellular Localization

TheDTH8ORF without a termination codon was cloned into the pA7-GFP

vector, placing the DTH8 gene upstream of the GFP coding sequence to create

in-frame fusion ofDTH8 cDNA and GFP reporter gene. The fusion constructs,

as well as the control (pA7-GFP), were transformed into onion (Allium cepa)

epidermal cells by particle bombardment. The transformed onion epidermal

cells were incubated at 22�C on Murashige and Skoog plates in the dark for

48 h. Then, the cells were examined under confocal fluorescence microscopy.

Wei et al.




Scanning Electron Microscopic Observation

Culms of Asominori and CSSL61 were harvested 10 d after flowering and

fixed in 2.5% v/v glutaraldehyde. Then, the samples were post fixed in 2%w/v

OsO4 for 2 h, dehydrated through an ethanol gradient, infiltrated, and

embedded in butyl methyl methacrylate. The samples conducted with critical

point dry and sputter coated with platinum and observed using a scanning

electron microscope (S-3000N, Hitachi).

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers HM775396 and HM775397.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. The flow chart of genetic study of DTH8 in this

article.

Supplemental Figure S2. Flowering times of Asominori and CSSLs

carrying the chromosome segments including DTH8 from the donor

parent IR24 with Asominori background under NLD conditions.

Supplemental Figure S3. Distribution of days to heading under NLD

conditions of two secondary F2 populations derived from the cross

between CSSL8, CSSL61, and Asominori.

Supplemental Figure S4. Sequence alignment of the conserved domains of

the HAP3 subunits from rice, Arabidopsis, yeast (ScHAP3, NP_009532),

and human (HsNF-B, L06145).

Supplemental Figure S5. The expressions of DTH8 and other flowering

genes in Asominori and CSSL61.

Supplemental Figure S6. Expressions of DTH8, OsGI, Hd1, Ehd1, Hd3a,

and Ghd7 in the cv Asominori and CSSL61 siblings during the diurnal

cycle.

Supplemental Figure S7. The expression of DTH8 and other flowering

genes in the NILs of Hd1 and Ghd7 under LD conditions.

Supplemental Figure S8. Plant height phenotype of cv Asominori and

CSSL61.

Supplemental Figure S9. DTH8 alleles of type 4 (from cv Kasalath), type

5 (from cv 93-11), and type 6 (from cv IR64) were expressed driven by

35S in CSSL61 (with nonfunctional allele from IR24) background.

Supplemental Table S1. DTH8 was mapped over 5 years in NLD condi-

tions based on a RIL population derived from two parents, cv Asominori

(japonica) and cv IR24 (indica).

Supplemental Table S2. Phenotypes of Asominori and CSSLs carrying the

chromosome segments including DTH8 from the donor parent IR24

with Asominori background under NLD conditions.

Supplemental Table S3. Phenotypes of Asominori and CSSL61 under NSD

conditions.

Supplemental Table S4. DTH8 alleles and its PS of flowering time among

40 rice varieties from a wide geographic range of Asia.

Supplemental Table S5. Phenotypes of transgenic and nontransgenic

segregants in the T1 generation derived from T0 plants with single-copy

gene grown under NLD conditions.

Supplemental Table S6. The sequences of primers used for mapping

DTH8.

Supplemental Table S7. Primers for vector constructions in this article.

Supplemental Table S8. Primers for QRT-PCR and semiquantitative

RT-PCR in this article.

ACKNOWLEDGMENTS

We are grateful to Professor A. Yoshimura for providing us the RIL and

CSSL populations and genotype data; Professor M. Yano for the NILs of Hd1;

and Professor Q.F. Zhang for the NILs of Ghd7.

Received March 28, 2010; accepted June 7, 2010; published June 21, 2010.

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DTH8 Suppresses Flowering in Rice, Inﬂuencing · PDF fileDTH8 Suppresses Flowering in Rice, Inﬂuencing Plant Height and Yield Potential Simultaneously1[W][OA] Xiangjin Wei, Junfeng