Positional cloning and characterization reveal themolecular basis for soybean maturity locus E1that regulates photoperiodic floweringZhengjun Xiaa,b,1, Satoshi Watanabeb,1, Tetsuya Yamadac, Yasutaka Tsubokurab, Hiroko Nakashimad, Hong Zhaia,Toyoaki Anaid, Shusei Satoe, Toshimasa Yamazakif, Shixiang Lüa, Hongyan Wua, Satoshi Tabatae, and Kyuya Haradab,1

aNortheast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150081, China; bSoybean Applied Genomics Research Unit,National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan; cGraduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;dFaculty of Agriculture, Saga University, Saga 840-8502, Japan; eDepartment of Plant Genome Research, Kazusa DNA Research Institute, Kisarazu 292-0812,Japan; and fBiomolecular Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan

Edited by Detlef Weigel, Max Planck Institute for Developmental Biology, Tübingen, Germany, and approved April 5, 2012 (received for review November10, 2011)

The complex and coordinated regulation of flowering has highecological and agricultural significance. The maturity locus E1 hasa large impact on flowering time in soybean, but the molecularbasis for the E1 locus is largely unknown. Through positional clon-ing, we delimited the E1 locus to a 17.4-kb region containing anintron-free gene (E1). The E1 protein contains a putative bipartitenuclear localization signal and a region distantly related to B3 do-main. In the recessive allele, a nonsynonymous substitution oc-curred in the putative nuclear localization signal, leading to theloss of localization specificity of the E1 protein and earlier flower-ing. The early-flowering phenotype was consistently observed inthree ethylmethanesulfonate-induced mutants and two naturalmutations that harbored a premature stop codon or a deletion ofthe entire E1 gene. E1 expression was significantly suppressed un-der short-day conditions and showed a bimodal diurnal patternunder long-day conditions, suggesting its response to photoperiodand its dominant effect induced by long day length. When a func-tional E1 gene was transformed into the early-flowering cultivarKariyutaka with low E1 expression, transgenic plants carrying ex-ogenous E1 displayed late flowering. Furthermore, the transcriptabundance of E1 was negatively correlated with that of GmFT2aand GmFT5a, homologues of FLOWERING LOCUS T that promoteflowering. These findings demonstrated the key role of E1 inrepressing flowering and delaying maturity in soybean. The molec-ular identification of the maturity locus E1 will contribute to ourunderstanding of the molecular mechanisms by which a short-dayplant regulates flowering time and maturity.

photoperiodism | quantitative trait locus | photoperiodic insensibility

The complex processes that control flowering are critical to howplants maximize their reproductive success, and therefore have

high ecological and agricultural importance. Plants use variousregulatory networks or pathways to respond to photoperiod andother environmental cues (1, 2). In 1920, Garner and Allarddemonstrated that soybean and several other plant species flowerin response to changes in day length and described this phenom-enon as “photoperiodism” (3). Deciphering genes involved inphotoperiodic pathways is the key to understanding the exquisitecoordination of various flowering processes. In Arabidopsis thali-ana and rice (Oryza sativa), a conserved pathway that confers daylength responses through regulation of FLOWERING LOCUS T(FT) transcription by CONSTANS (CO) has been well charac-terized (4–7). The FT protein is a main component of florigen,which is a mobile flowering-promotion signal produced in leavesand transported through the phloem to the meristems (8–12). InArabidopsis, CO directly induces FT expression under long-dayconditions (4). Despite the conserved functions ofFT homologues,the regulation and expression of FT may vary in different plantspecies (13, 14). Research on morning glory (Pharbitis nil) (14, 15)and tomato (Solanum lycopersicum) (13, 16) showed that theregulatorymechanism for flowering time could be different even in

plants that exhibit the same overall response to day length. This isbecause the day length response can diverge rapidly during evo-lution (17).Soybean is a valuable plant species for studying photoperiodic

effects on flowering because of its typical short-day flowering,widespread cultivation, and agricultural importance. Floweringtime and time to maturity in soybean are important quantitativetraits related to photoperiod adaptability, domestication, andproductivity. As early as the 1920s, researchers started to identifygenetic factors controlling flowering and maturity in soybean. In1927, Owen detected a major pair of genes controlling maturityand designated them as E and e (18). In 1971, Bernard concludedthat these genes were the same asE1 and e1, two alleles of a singlemajor locus affecting maturity in his study (19). The E1 locus islargely responsible for the variation in flowering time amongsoybean cultivars (19, 20). To date, eight flowering time or ma-turity loci, designated E1 to E8 (19, 21–27), along with the J locusfor “long juvenile period” (28), have been genetically identified.Of these, E1, E3, and E4 are involved in photoperiod responses(20–23, 29, 30).To date, three of the maturity loci have been molecularly

identified. E3 and E4 encode GmPHYA3 (31) and GmPHYA2(32), respectively, which are homologues of the photoreceptorphytochrome A (PHYA) (33). E2 encodes a homologue ofGIGANTEA (34), a nuclear-localized membrane protein thatfunctions upstream of CO and FT in A. thaliana (35). In addition,homologues of many other Arabidopsis flowering-time genes arepresent in soybean (36, 37). Two functionally coordinated soy-bean GmFT genes (GmFT2a and GmFT5a), the homologues ofthe Arabidopsis FT, are responsible for inducing flowering undershort-day conditions and are likely to be involved in the phyto-chrome A signaling pathway (38). Functional phytochrome Agenotypes suppressed the expression of GmFT2a and GmFT5aunder long-day conditions and delayed flowering, whereas dou-ble-recessive PHYA genotypes induced GmFT expression andpromoted early flowering regardless of day length (38).

Author contributions: Z.X. and K.H. designed research; Z.X., S.W., T. Yamada, Y.T., H.N.,H.Z., T.A., S.S., S.L., and H.W. performed research; T. Yamazaki and S.T. analyzed data;and Z.X., S.W., and K.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the DNAData Base in Japan, http://www.ddbj.nig.ac.jp (DDBJ accession nos. AB552962, AB552963,AB552971, AP011812, AP011814–AP011820, and AP011823).1To whom correspondence may be addressed. E-mail: xiazhj@neigaehrb.ac.cn, nabemame@affrc.go.jp, or haradaq@nifty.com.

See Author Summary on page 12852 (volume 109, number 32).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1117982109/-/DCSupplemental.

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Thakare et al. compared the transcriptional profiles of homo-logues ofArabidopsis flowering-time genes between soybean near-isogenic lines (NILs) harboring contrasting E1 alleles (39). Al-though several genes, including the homologues of CO, displayeddifferent expression patterns under long- and short-day con-ditions, they did not differ noticeably in their expression patternsbetween the E1 NILs (39, 40), except that the abundance ofGmFT transcripts was negatively correlated with flowering timeunder long-day conditions (40). The regulatory network control-ling photoperiod response and flowering time through photore-ceptor genes (E3, E4) and GmFT genes in soybean thereforeremains largely unknown because of the lack of understanding ofthe key gene at the E1 locus.The association between gray pubescence color (t/t) and late

maturity (E1/E1) was first perceived in 1927 (18), and confirmed byrecombination rates from 1.4% to 4.0% (19, 26). The E1 locus waslinked to the Satt365 marker in linkage group (LG) C2, on chro-mosome 6 (Gm06) (26, 41). Based on the soybean genome se-quence (42) (http://www.phytozome.net), the E1 locus resides ina pericentromeric region. Such regions are repeat-rich and gene-poor, with a high ratio of physical to genetic distance. This makes itdifficult to precisely locate and characterize pericentromeric genes.As a result of its photoperiodic responsiveness, the E1 gene

was previously assumed to be a member of the GmPHYB family(36). However, this became implausible when none of theGmPHYB members were genetically or physically mapped inproximity to the E1 locus (36, 42). Therefore, we focused ondeciphering the molecular basis of the E1 locus through posi-tional cloning in the present study. By using a large population,we delimited the E1 locus to a 17.4-kb region containing a singlegene. Our functional characterization of natural variation incultivars and in ethylmethanesulfonate (EMS)-derived mutantsvalidated the molecular identity of the E1 gene. Moreover,transcriptional profiling of E1 under different environmentalconditions and in various genetic backgrounds enabled us to es-tablish a preliminary genetic model for photoperiodic floweringpathway in soybean.

ResultsE1 Locus Was Delimited to a 17.4-kb Region That Contained a SingleGene. Mapping populations were originally derived from a crossbetween two E1 NILs, Harosoy-E1 (E1e2E3E4e5) and Harosoy(e1e2E3E4e5), which carry contrasting E1 alleles. Harosoy-E1required 45.0 ± 0.78 d (mean ± SD) to reach R1 (from emergenceto opening of the first flower) stage (43), which was 10 d longerthan Harosoy (e1) (34.9 ± 0.83 d) under natural field conditions atMatsudo, Japan (35°78′N, 139°90′E), in 2005. The E1 allele ispartially dominant over e1, because F1 plants of the cross betweenthese lines flowered at 41.5± 1.16 d after emergence. TheE1 locuswas initially mapped close to marker Satt557, which is betweenmarkers Satt365 and Satt289, by means of quantitative trait locusanalysis of flowering time in an F2 population (117 plants) atMatsudo in 2005. Seven recombinants carrying a crossover be-tween markers Satt365 and E5 were identified in 2006 in an F2:3population consisting of 1,442 individuals derived from 51 F2plants that were heterozygous at Satt557 (Fig. 1 and Fig. S1). TheE1 genotype of each recombinant was determined based on itsflowering time in 2006, and was confirmed based on the segrega-tion pattern among its progeny in 2007 (Fig. S1 and Table S1) atTsukuba, Japan (36°03′N, 140°04′E). No recombination was foundbetween themarkers S8 and Satt557, despite a physical distance of133 kb (Fig. 1). This might be a result of the low recombinationrate that often occurs in the pericentromeric region (Fig. 1) (42).At this stage of the mapping study, we were able to delimit the E1region to an interval of ∼289 kb between the markers A and E5(Fig. 1). However, given that more than 40 genes were predicted inthis 289-kb region using RiceGAAS (44), more intensive fine-scalemapping was conducted. With a simple manual protocol (SIMaterials and Methods) for large-scale genotyping of soybeanseeds, we successfully screened 13,761 F2:5 seeds harvested fromself-crossed F2:4 plants carrying a heterozygous E1 segment, andidentified 10 recombinants carrying crossovers within the 289-kb

region (Fig. 2A). The E1 genotype of each recombinant was de-termined by evaluating the phenotypic segregation pattern of theprogeny at Tsukuba in 2009 (Fig. S1 and Table S1). The differ-ences in flowering time (R1) among the genotypes were statisti-cally significant (ANOVA, P < 0.001; Table S1).The E1 locuscosegregated with the markers 34 and TI in these recombinants,thus delimiting the candidates for theE1 gene to a region betweenmarker 33 and marker 12.In two physical contigs (Fig. S2) built from two independent

BAC libraries, the delimited region respectively corresponds to17,372 bp for the dominant E1 allele in Misuzudaizu and 22,876bp for the recessive e1 allele in Williams 82 (Fig. 2 B and C). Asingle intron-free gene (AB552962, 525 bp, 174 aa) was consis-tently identified by GenScan (45), GeneMark (46), FGENESH(http://linux1.softberry.com/berry.phtml), and RiceGAAS (44)for the dominant E1 genotype (Misuzudaizu and Harosoy-E1),and was designated E1 (Fig. 2D). In Williams 82 (39) and Har-osoy (e1), which carry the recessive e1 allele, a single missensepoint mutation occurred at nucleotide 44 in the coding region ofE1 (Fig. 2D), leading to a substitution of threonine for arginineat amino acid residue 15. We designated this recessive allele ase1-as (AB552963). We also detected 14 SNPs or indels (2–3 bp)between E1 and e1-as in the promoter regions. Moreover, aninsertion of a 5,537-bp retrovirus-related sequence, within whicha gag/pol protein was predicted by RiceGAAS (44), was insertedin the region 4 kb upstream of e1-as (Fig. 2C), although it is notclear whether this insertion has any functional impact on e1-as.The predicted e1-as protein (174 aa) corresponds to Gly-ma06g23040.1 (149 aa) in the Williams 82 genome (42) (http://www.phytozome.net), indicating a difference in gene prediction.Our RACE PCR analysis supported our prediction: the full-

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Fig. 1. Genetic mapping of the E1 locus. The mapping population wasdeveloped by crossing the NILs, Harosoy-E1 and Harosoy (e1). The E1 locuswas initially delimited to a 289-kb region (in red) in the pericentromericregion of LG C2 (chromosome Gm06) in the soybean genome (http://www.phytozome.net) from 2006 to 2007. No recombination occurred betweenmarkers S8 and Satt557 despite the 133-kb physical distance, indicating a lowrecombination rate in the E1 region. The twisted arrows represent re-combination.

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length cDNA sequence of E1 was 838 bp, with 157 bp and 156 bpfor the 5′ and 3′ UTRs, respectively, in Harosoy-E1 and Harosoy(e1) (AB552964 and AB552965; Fig. 2D).

Allelic Variation in E1 Associated with Flowering Time. Sequencesfrom soybean cultivars with different flowering times were eval-uated (Table S2) to identify association between flowering timeand allelic variations in E1, and this analysis led to the identifi-cation of two additional recessive alleles (Fig. 2E) from the early-flowering cultivars. One allele, designated e1-fs (AB552971), hada 1-bp deletion in codon 17 that resulted in a premature stopcodon in Sakamotowase and its derived NILs (20). The second,designated e1-nl, was a null allele in which ∼130 kb (includingthe entire E1 gene) was deleted in some early-flowering cultivars[i.e., Fiskeby V, Yukihomare (Table S2), Toyosuzu, Toyomusume,Hejian 1, and Heihe 28] known for photoperiod-insensitivity orbeing cultivated at high latitudes. The absence of the E1 sequencein these cultivars was confirmed by Southern hybridization (SIMaterials and Methods and Fig. S3).To evaluate the flowering time for the differentE1 genotypes, we

grew the cultivars in a growth chamber under long-day conditions(16 h light/8 h dark). Cultivars with E1 (except the E1/E2/e3/e4genotype, with R1 of 36 d), e1-as, e1-fs, and e1-nl genotypes flow-ered∼70,∼50,∼30, and∼30 d after emergence, respectively (TableS2). The e1-fs and e1-nl genotypes showed a similar early-floweringphenotype, with no apparent delay in flowering time under long-

day conditions, despite different genetic compositions at the otherE loci (Table S2), suggesting the truncated e1-fs–produced proteinis nonfunctional. Cultivars with the e1-as genotype generally hadflowering times intermediate between the E1 and e1-fs genotypesboth in the growth chamber and in the field, suggesting that e1-as isa leaky allele and may retains partial E1 function. The functionaldifferences among the e1 alleles observed in the present study areconsistent with a recent report (47) in which a flowering-time QTLat the E1 locus was detected in a population derived fromHarosoy-e3 (e1-as) and Sakamotowase (e1-fs).

EMS-Derived E1 Mutants Show Early-Flowering Phenotype. To con-firm the function of E1 in delaying flowering, we identified mutantlines from EMS-treated libraries by using the Targeting InducedLocal Lesions IN Genomes approach (48). We obtained three in-dependentE1mutants withmissensemutations (Fig. S4), i.e., serineto phenylalanine at aa 17 (e1-m1), arginine to lysine at aa 15 (e1-m2), and threonine to isoleucine at aa 65 (e1-m3). The e1-m1 mu-tation was generated in OLERICHI50, whereas e1-m2 and e1-m3were generated in Fukuyutaka. Both WT OLERICHI50 andFukuyutaka carry the E1 allele, yet they have different floweringtimes (Table 1), which is most likely because of differences in theirgenetic backgrounds (20, 30). All three mutant lines flowered sig-nificantly earlier compared with their respective WT plants (Table1). In addition, the line with e1-m1/e1-m1 flowered at 33.7 ± 0.47 d(n= 14), vs. 41.6± 1.8 d (n= 13) for theWT (OLERICHI50) under

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Fig. 2. Positional cloning and characterization of the E1 locus. (A) Graphical genotypes of 10 recombinants carrying crossovers in the E1 region. The E1genotype of each recombinant was confirmed based on the phenotypic segregation pattern in its progeny (Fig. S1, Right). The delimited region for the E1locus is shaded in pink. (B) The corresponding marker positions in physical contigs of Williams 82 and Misuzudaizu. (C) Within the delimited region inMisuzudaizu, a single intron-free gene (red) was identified. A 5,537-bp retrovirus-related sequence (yellow) containing a 1,401-bp putative gag/pol retrovirusgene (green) was inserted ∼4 kb upstream of e1-as in Williams 82. (D) Genetic variation in the E1 allele between Harosoy-E1/Misuzudaizu and Harosoy (e1)/Williams 82. The UTR and coding regions of E1 are marked in blue and red, respectively. (E) Apart from the dominant E1 gene, we identified three recessive e1genotypes (as, missense point mutation; fs, frameshift mutation; nl, null mutation).

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natural field conditions at Tsukuba. Under long-day conditions, theflowering times of F2 plants derived from a cross between the ho-mozygous e1-m1 mutant and the WT were significantly correlatedwith the genetic mutation at the E1 locus (P < 0.01; Table 1). Themutant lines, e1-m2/e1-m2 and e1-m3/e1-m3, both flowered signif-icantly earlier than the WT (Fukuyutaka; Table 1). Similarly, thephenotypes of the progeny of the self-pollinated heterozygousgenotypes (E1/e1-m2 or E1/e1-m3) were significantly correlatedwith the genetic mutations at the E1 locus under natural conditions(P < 0.05; Table 1). The similar phenotype for three independentmutant lines and the significant differences in flowering time amonggenotypes in the mutant-derived progenies indicated that themutations at E1, rather than at other loci, were responsible for al-ternation of the phenotype. Taken together, the results obtainedfrommap-based cloning and the genetic and phenotypic analysis ofthe natural and artificial mutants strongly supported the hypothesisthat E1 is the gene governing flowering time at the E1 locus.

In Silico Analysis and in Vivo Subcellular Distribution. Two E1paralogues (E1-L genes)Glyma04g24640.1 andGlyma18g22670.1are present in the genome of soybean variety Williams 82, andeach has 94% nucleotide sequence identity to E1 (Fig. 3A). Thisprediction was supported by the results of Southern hybridization(Fig. S3). However, in contrast to the annotation for Gly-ma18g22670.1 (156 aa; http://www.phytozome.net), this gene waspredicted to encode 173 aa by RiceGAAS, and was referred to asGm18g22670 in the present study (Fig. 3 A and B). Two othergenes, Glyma10g17480.1 and Glyma10g20520.1, shared moderate(56.8% and 52.7%, respectively) amino acid sequence identity tothe E1 protein (Fig. 3B). In legumes, sequences highly homolo-gous to E1 were retrieved from Phaseolus vulgaris, Medicagotruncatula, and Lotus japonicus (Fig. 3 A and B).No known conserved domain was present in the E1 protein (E <

0.01) based on primary sequences in the National Center for Bio-technology Information Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). An analysis using I-TASSER (49) and Phyre (50) software showed that the E1 proteinshared a low level (21–27%) of amino acid sequence identity withB3 domain sequences. The B3 domain has been identified ina number of transcriptional factors specific to plant species (46)(Fig. S5 A and B). Interestingly, the C-terminal region (aa 55–174)carries the minimal B3 domain structure required for DNA contact(51, 52) (Fig. S5A). In fact, there are several genes that contain a B3domain in the soybean genome (Fig. 3B), although their functionshave not been well characterized. In addition, prediction of the 3Dstructure using I-TASSER also showed a helix–turn–helix structurefor the N-terminal region (aa 1–54; Fig. 3C). More importantly,a putative bipartite nuclear localization signal (NLS) with basicdomains at each end (KKRK and RRR), separated by 12-aa resi-dues, was identified at aa 13 to 31 (aa 15–33 in the consensus se-

quence; Fig. 3A). The features identified by in silico analysisindicated that the E1 protein may function in DNA binding or asa transcription factor.To gain further knowledge regarding the function of the E1

protein, we conducted a transient expression assay to determine itssubcellular distribution. We separately fused E1, e1-as, and e1-fs tothe eGFP gene and obtained constructs P35S:E1-eGFP, P35S:e1-as-eGFP, and P35S:e1-fs-eGFP, respectively. Confocal imagingshowed that the E1-eGFP fusion protein was distributed primarilyin the nucleus, with little signal in the cytoplasm either in A.thaliana protoplasts (Fig. 4A) and in onion (Allium cepa) epider-mal cells (Fig. 4B). In contrast, the e1-as-eGFP fusion protein wasdistributed in the nucleus and the cytoplasm of both cell types (Fig.4 A and B). There was no signal for the e1-fs:eGFP construct(Fig. 4A), indicating that the predicted protein (corresponding toaa 55–174 of the E1 protein) was not produced as a result of theframeshift mutation (e1-fs). This result indicated that an aminoacid substitution in the basic domain of the putative bipartite NLSaffected nuclear targeting of the E1 protein (53, 54). The changesin subcellular localization may represent a key mechanism un-derlying difference in flowering time governed by E1 and e1-as.

Transcriptional Profiling of E1. To examine how E1 transcriptionlevels are related to flowering time, we analyzed the expressionpatterns of E1 and other related genes under different day lengthconditions. The low in planta abundance of E1 transcripts(demonstrated by RT-PCR) may explain the lack of EST data forE1 in the GenBank database (55, 56). BE608878, the only se-quence we found in GenBank might have originated from Gly-ma04g24640.1, the E1 paralogue, as they are almost identical(405 of 406 bp identity). The expression of E1 was tissue-specific,with high levels in fully expanded leaves (including cotyledons)and low levels in other tissues (Fig. 5A).The expression of the clock-gene homologueGmLCL2 (Glycine

max LHY/CCA1 Like2) (39, 57) exhibited a circadian rhythm (Fig.5B), with no noticeable difference between short- and long-dayconditions or between the two NILs (Fig. 5C), which was similar toprevious observations (39). In contrast, the transcript abundancesof E1 in both Harosoy NILs were low under short-day conditions(12 h light/12 h dark; Fig. 5 B and C). Under long-day conditions(16 h light/8 h dark), diurnal expression of E1 showed a bimodalpattern (Fig. 5 B and C). In the dark, E1 transcription graduallydecreased, reaching its minimum before dawn. E1 transcriptionappeared to be reset at dawn and dusk (i.e., twice per day). Thehigh level of suppression under short-day conditions and the bi-modal pattern under long-day conditions suggested that the ex-pression of this gene was regulated by photoperiod. Further studyis needed to reveal whether the photoperiodic regulation pathwaysare entangled with the circadian clock. Interestingly, two E1-Lgenes (Glyma04g24640.1/Gm18g22670) showed an expression

Table 1. Phenotypic analysis of flowering time in the WT and the progenies of E1 mutants (mut) obtained using EMS mutagenesis

Mutant lineMutation inE1 protein‡ Population

Flowering time, days after emergence

P value§WT/WT WT/mut mut/mut

E1-m1* Ser17Phe Homozygous mut × WT, F2 69.70 ± 1.37 (12) 66.80 ± 1.82 (12) 63.80 ± 1.07 (12) < 0.001E1-m2† Arg15Lys Heterozygous mut, self-crossed 47.67 ± 1.25 (9) 45.38 ± 2.15 (16) 38.71 ± 1.03 (7) < 0.001

Arg15Lys Homozygous mut — — 38.11 ± 1.73 (9) —

E1-m3† Thy65Ile Heterozygous mut, self-crossed 46.75 ± 1.92 (4) 43.75 ± 1.64 (8) 37.33 ± 2.05 (3) 0.01 < P < 0.05Thy65Ile Homozygous mut — — 35.20 ± 1.72 (5) —

WT Normal WT, Fukuyutaka 48.57 ± 0.90 (7) — — —

—, not applicable or not available.*The mutant (mut) line was derived from OLERICHI50. The F2 population had a ratio of 18:47:22 for homozygous WT, heterozygous, and homozygous mutantalleles, respectively, and was thinned to an equal number of plants (n = 12) of each genotype for phenotypic investigation under artificial long-day conditions(15 h light/9 h dark for 30 d after sowing and thereafter 14 h light/10 h dark).†These mutant lines were derived from Fukuyutaka. The progenies of plants harboring heterozygous or homozygous mutant alleles were grown undernatural day length conditions [14:33 (h:min) at emergence to 13:23 at R1 for Fukuyutaka]. WT plants (Fukuyutaka) were grown as a control.‡The sequence electrograms for representative mutant lines are shown in Fig. S4.§The statistical significance of the phenotypic differences within each population was evaluated using one-way ANOVA.

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pattern similar to E1 (Fig. 5B), suggesting that these genes mightbe regulated by a similar mechanism.Under short-day conditions, the transcriptions of GmFTs

(GmFT2a and GmFT5a), two homologues of Arabidopsis FT(38), were strongly activated in soybean NILs carrying E1 or e1-as, with peak transcription occurring shortly before sunrise, fol-lowed by a steady decline until the end of the light period (Fig.5C). Under long-day conditions, the overall transcription of thetwo GmFTs was lower than that under short-day conditions.Intriguingly, GmFTs in the leaf tissue of the e1-as genotype wereexpressed at significantly higher levels than that of the E1 ge-notype, peaking shortly after sunrise, followed by a steady de-cline until noon, and then increasing again to form a minor peakshortly after sunset (Fig. 5C). Given that there were no signifi-cant differences in transcript abundance between E1 and e1-as(Fig. 5 B and D), the defect of function in e1-as might be a resultof the point mutation at the protein level.

Other genetic factors might also influence E1 expression. Totest this possibility, we analyzed E1 transcription levels by usingNILs with different genetic backgrounds. In previous research,E1 and the photoreceptor (i.e., PHYA) genes E3 and E4responded differently to photoperiod (20, 30). Even under longdays, E1 expression was significantly suppressed in NIL 130E(Fig. 5E) and in Kariyutaka (58), both of which carry the double-recessive alleles e3 and e4. This is consistent with previousobservations that cultivars carrying either e1 or the double re-cessive alleles (e3 and e4) showed decreased sensitivity to pho-toperiod (20, 30, 38). Transcriptional profiling in the presentstudy indicated that a high level of E1 expression requires long-day conditions and functional PHYA genes.

Elevated Expression of E1 Resulted in Late Flowering in TransgenicSoybean. Transgenic plants with high expression of exogenousE1 were evaluated to further characterize the function of the E1

*.* * * *:**** .: *****: .* .::: : .**********GM18g22670 MSNHSD--EKEQCQKKRKSTICEASNFRTSRRRFCSN-KNEEEMN-------KGVSTTLKLYDD 54Gma_Glyma04g24640.1 MSNPSD--EKEQCQKKRKSTICEASNFKTSRRRFFSN-KNEEDMN-------KGVSTTLKLYDD 54Pvu_23533527 MSNPGD--EKELCQKKRKSTICEASNFRTSRRRFCSN-QSEEEMN-------KGVSTTLKLYDD 54E1_gene MSNPSD--EREQCQKKRKSTICEASNFRTSRRRFCSNNKNEEEMNN------KGVSTTLKLYDD 56LJ1_chr5.CM0328.440.r2.d MNNLGD--ETEFCQRKRKSPSSEGS---TSRRRFSSN--NNNEKD--------GVSTTLKLYDD 49Mtr_Medtr2g073930.1 MNNIHLRVEMEQLQKKRKSCDEASTNLKTSRRRLCNN-KNEEQNNNQNNDNKGSVSTTLKLYDD 63

1.......10........20........30........40........50........60....

******:* ******.** **:***********.. .****** :*: *.***::*:***:**GM18g22670 PWKIKKTLTDSDLGILSRLSLATDLVKKQILPMLGADHARAAETEEGTPVRVWDMDTKSMHQLV 118Gma_Glyma04g24640.1 PWKIKKTLTDSDLGILSRLSLAADLVKKQILPMLGADHARAAETEEGTPVRVWDMDTKSMHQLV 118Pvu_23533527 PWKIKKTLTDSDLGILSRLLLAADLVKKQILPMLGADHARAAETEEGTPVRVWDIDTKSMHQLV 118E1_gene PWKIKKTLTDSDLGILSRLLLAADLVKKQILPMLGAYHARAAET-EGTPVRVWDMDTKSMHQLV 119LJ1_chr5.CM0328.440.r2.d PWKIKKTLMASDLGILNRLMLAADLVKKQILPMLGVHQARAAETEQGSQVRVWDVDTESMHQLV 113Mtr_Medtr2g073930.1 PWKIKKSLTESDLGILSRLLLAADLVKKQILPMLDVDDARAAETEEGSPVNVWDMETNSMHELV 127

....70........80........90.......100.......110.......120.......1

*************.**.*******:*:******* ***:* *******:* :.GM18g22670 LKRWSSSKSYVLIAKWNQDFVRRRDLKKGDEIGFHWDPYNCVFNFCVLKRA--------MPEN 173Gma_Glyma04g24640.1 LKRWSSSKSYVLIGKWNQDFVRRRDLKKGDEIGFHWDPYNCVFNFCVLKRA--------MPEN 173Pvu_23533527 LKRWSSSKSYVLIGKWNQDFVRRRDLKKGDEIGFHWDPYNCIFNFCVLKRA--------MPEN 173E1_gene LKRWSSSKSYVLIGKWNQDFVRRRDLRKGDEIGFHWDPYNCVFNFCVLKQA--------MPEN 174LJ1_chr5.CM0328.440.r2.d LKRWSSSKSYVLIGKWSQDFVRRRELKKGDEIGFYWDPYNCAFNFCVLKRARSLGLDHLMSHD 176Mtr_Medtr2g073930.1 LKRWSSSKSYVLIGKWNQDFVRRRELKKGDEIGFQWDPFNRAFNFCVLKRA--------IPP- 181

30.......140.......150.......160.......170.......180.......190.

A

B CE1 geneGlyma04g24640.1GM18g22670Pvu 23533527LJ1 chr5.CM0328.440.r2.dMtr Medtr2g073930.1LJ2 chr6.CM0830.560.r2Ccl 19275809Csi 18094876Glyma10g17480.1Glyma10g20520.1Mtr Medtr1g059640.1Mtr Medtr1g065720.1Mtr Medtr1g065730.1Mtr Medtr1g065750.1Cpa 16427529Egr 23578593Ptr 18216763Mes 17977842Rco 16801902Mx 22627980Bra 22691616Cru 20899217Aly 891945Aco 22049220Lus 23151243Tha 20198438Mgu 17678668Aly 347288At AT2G33720Ppe 17650946Zm 20852057Os VP1Ta VP1At AP2Glyma20g39140.1Os B3Zm RAV1At TEM1Glyma16g01950.1Glyma10g34760.1Glyma20g32730.1Glyma01g22260.1Glyma02g11060.1

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Fig. 3. Characterization of the E1 protein. (A) Alignment of sequences highly homologous to E1 from legumes (Glyma or GM, G. max; Lja, L. japonicus; Mtr,M. truncatula; Pvu, P. vulgaris). The putative bipartite NLS is underlined, with its two basic domains (KKRK and RRR) marked by red squares. (B) A phylogenetictree of E1 and its homologous sequences from various plant species. Sequences highly homologous to the E1 protein have a blue background. The sequencescontaining a standard B3 domain have a green background. Gm18g22670 was predicted by RiceGAAS (41) to have 173 aa, and is 17 aa longer than (butcorresponds to) Glyma18g22670.1 (http://www.phytozome.net). Predicted protein sequences of Lja_chr5.CM0328.440.r2.d and Lja_chr6.CM0830.560.r2 wereretrieved from http://www.kazusa.or.jp/lotus/blast.html. Other protein sequences were mainly retrieved from phytozome.net (version 8.0). Aly, Arabidopsislyrata; At, A. thaliana; Bra, Brassica rapa; Ccl, Citrus clementine; Cpa, Carica papaya; Cru, Capsella rubella; Csi, Citrus sinensis; Egr, Eucalyptus grandis; Lja, L.japonicus; Lus, Linum usitatissimum; Mgu,Mimulus guttatus; Mtr,M. truncatula; Os, O. sativa; Pvu, P. vulgaris; Tae, Triticum aestivum; Zm, Zea mays. The genename or reference number at phytozome.net is indicated after the species abbreviation. Typical B3-domain sequences obtained from the GenBank database(http://www.ncbi.nlm.nih.gov) are from A. thaliana, At AP2 (NP_175483.1) and At TEM1 (NP_173927.1); from O. sativa, Os B3 (EAY75457.1) and Os VP1(Os01g0911700); from T. aestivum, Tae VP1 (CAB91107.1); and from Z. mays, Zm RAV1 (NP_001141742.1). Their homologues in soybean were also included forcomparison. (C) A 3D structural model of the E1 protein generated by using the I-TASSER software (49), which predicts a B3-like fold for the C-terminalresidues (aa 55–174, pink) and a helix–turn–helix structure for the N-terminal region (blue). Side chains of I71, L72, A79, W123, S124, S125, and K127 thatcorrespond to the predicted DNA-contacting residues of AtRAV1 are shown in the ball-and-stick representation. In the e1-as protein, Arg is replaced by Thr atthe position of the R15 side chain.

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protein. The early-flowering Japanese cultivar Kariyutaka (E1/e3/e4) showed low levels of E1 expression under natural fieldconditions at Tsukuba and under long-day conditions in a growthchamber with light provided by white fluorescent lights supple-mented by incandescent light. Because Kariyutaka is highlyamenable to Agrobacterium-mediated transformation (58) andexpresses E1 at extremely low levels (Fig. 6), this cultivar wasselected for transformation of E1 under its natural promoter totest whether the E1 expression level was associated with theflowering phenotype. A 4,332-bp fragment (AB552966) con-taining a 2,668-bp upstream sequence from the start codon, theE1 coding region, and a 1,139-bp downstream sequence from thestop codon was amplified from Harosoy-E1 and cloned intothe pMDC123-GFP vector (58). We characterized nine trans-genic T1 plants derived from three independent T0 plants underlong-day conditions (16 h light/8 h dark). In six transgenic plants(derived from two different T0 plants) that harbored one or twocopies of the transgene, higher levels of E1 transcripts in theleaves were observed compared with the WT or vector control(Table S3). In the same cDNA samples, lower levels of GmFTtranscripts were detected (Fig. 6 A and B). As expected, all sixplants displayed much later flowering than the WT and thevector control (Fig. 6C). Conversely, very low levels of E1 tran-script abundance coupled with high levels of GmFT transcriptionwere observed in two plants that carried seven or more copies ofthe transgene, and both showed early flowering (Fig. 6 A and Band Table S3). One plant with three copies of the transgenedisplaying relatively high E1 transcription and low GmFT tran-scription exhibited an intermediate flowering time (Table S3).Because high levels of E1 transcripts were always associated withlower GmFT transcription and later flowering, we hypothesizethat E1 regulates flowering time through its repression of GmFTtranscription, at least partially, under long-day conditions.

DiscussionFunction of the E1 Protein.Our in silico analysis suggests that E1 isamong a clade of sequences distantly related to the genes thatencode the plant-specific B3 domain. The B3 superfamilyencompasses many well characterized families and some poorlyunderstood ones with diverse functions in plant growth and de-velopment (52). Well characterized functions are available forthe LAV family [LEAFY COTYLEDON2 (LEC2)-ABSCISICACID INSENSITIVE3 (ABI3)-VAL], the ARF (AUXIN RE-SPONSE FACTOR) family, the RAV (RELATED TO ABI3and VP1) family, and the REM (REPRODUCTIVE MERI-STEM) family (52). Some genes that encode the B3 domain areinvolved in the control of flowering. Ectopic expression ofOsLFL1 (O. sativa LEC2 and FUSCAS3 Like 1) in the LAVfamily resulted in late flowering via suppression of Ehd1 ex-pression by binding to its promoter (59). TEMPRANILLO genes(TEM1 and TEM2) in the RAV family directly suppress FT genesin Arabidopsis (60), in which the quantitative balance between theactivator CO and the suppressor TEM determines the expressionlevels of FT. Mochida et al. (61) performed in silico analysis oftranscription factor repertories in soybean and deposited theirdata in LegumeTFDB (http://legumetfdb.psc.riken.jp) (62). Inthis database, the categories that include a B3 domain (RAV andARF) contain more than 230 genes, including homologues ofAtTEM genes (e.g., Glyma01g22260.1, Glyma02g11060.1, Gly-ma20g32730.1, and Glyma10g34760.1; Fig. 3B). However, wefound no information on E1 or its two paralogues in this database.E1 (corresponding to Glyma06g23040.1; http://www.phytozome.net) was classified into a “hypothetical gene family” at the Rosidnode, and into a “domain of unknown function (DUF313) fam-ily” at the Angiosperm node (http://www.phytozome.net; version8.0). We could not predict the function of E1 based on its lowsimilarity (21–27% at the amino acid level) to TEM1 or any otherwell characterized B3 genes (Fig. 3B and Fig. S5B). However,homologues of E1 from model legumes have not yet been char-acterized in vivo.Although our knowledge of the E1 protein is limited, we

obtained evidence of nuclear localization. Previous research

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Fig. 4. Subcellular localization of eGFP and of the E1-eGFP, e1-as-eGFP, ande1-fs-eGFP fusion proteins. (A) Arabidopsis protoplasts were transformed withplasmids that express the indicated gene constructs under the control of theconstitutive 35S cauliflower mosaic virus (CaMV) promoter and the nopalinesynthase terminator (T-Nos). The unfused eGFP coding sequence served asa control. The eGFP fusion proteins (E1-eGFP, e1-as-eGFP, and e1-fs-eGFP)were produced in the protoplasts and observed under a confocal laser-scan-ning microscope. GFP fluorescence, chlorophyll fluorescence, and bright-fieldimages, and overlays of the green and red images (“merged”), are shown. (B)Subcellular distribution of the E1 and e1-as proteins in onion epidermal cells.E1-eGFP and e1-as fusion proteins (or eGFP alone as a control) were producedtransiently under the control of the CaMV 35S promoter and T-Nos in onionepidermal cells and were observed under a confocal microscope. The photo-graphs were taken with a dark field for green fluorescence (Left), witha bright field for the cell morphology (Center), and with a combination ap-proach (merged, Right). Plasmolysis was performed to test for signals from thecell wall. In A and B, each 10 transformants were observed, and the experi-ments were performed three times, with similar results obtained.

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demonstrated that both basic domains in the bipartite NLS areessential for nuclear targeting (54), particularly at the position ofthe arginine residue in the first basic domain (63). In contrast,intervening amino acids can tolerate some point mutations andinsertions without any functional change (54). In the presentstudy, the point mutation from arginine to threonine at position15 in E1 (Fig. 3A) occurs at exactly the first basic domain ofKKRK (which changes to KKTK) in the bipartite NLS, leading todifferent subcellular distributions between E1 and e1-as (Fig. 4).Likewise, the change from arginine to lysine at the same positionin the e1-m2 mutant might also affect nuclear localization, al-though this hypothesis was not validated in vivo. The replacementof a highly conserved serine residue next to the NLS by aspartate,which mimics phosphorylation, might also result in decreasednuclear import (64). Similarly, the serine to phenylalanine mu-tation in e1-m1 occurs at aa 17, the position (Fig. S4A) next to thefirst basic domain, which might lead to changes in the phos-phorylation state, and thus in flowering time. In addition, thereplacement of threonine by isoleucine at aa 65 in the e1-m3mutant (Fig. S4A) occurred in a β-strand of the B3 domain that isconserved among several B3 proteins (Fig. S5), although thefunctional mechanism is not yet clear. The presence of a putativeDNA-binding B3 domain and a helix–turn–helix structure witha bipartite NLS suggests that the E1 protein might function asa transcription factor; however, further analysis is needed to re-veal the functional mechanism by which the E1 protein regulatesflowering time.Genomewide duplications occurred ∼59 and 13 Mya in soy-

bean (42). The high levels of sequence similarity at aa level amongthe E1 and E1-L genes indicate that these genes might haveresulted from a lineage-specific duplication in soybean. Intrigu-ingly, the expression pattern of E1-L in Harosoy-E1 is similar tothat of E1 (Fig. 5 A, B, and D). However, the physiologicalfunction of the E1-L genes in soybean needs to be clarified. Toour knowledge, no QTLs for flowering time have been locatedat either of the E1-L anchored regions (http://www.comparative-legumes.org) (65). Because the putative bipartite NLS and helix–turn–helix structure in the N-terminal region of the two E1-Lproteins are almost identical to those of E1, the eight amino acidchanges or indels in the C-terminal region (aa 31–173) mightresult in a subfunctionalization between E1 and E1-L.

Natural Variation in E1. The latitudinal distribution of a plant ora cultivar is strongly associated with its photoperiod sensitivity.In rice, polymorphism in Hd1 protein, the type of Hd3a pro-moter and the level of Ehd1 expression were related to diversityof flowering time and geographic adaptation in cultivated ricespecies (66). Ebana et al. (67) found that functional poly-morphisms in several identified key genes (Hd1, RFT1, andGhd7) contributed greatly to the diversity of rice heading dates.

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Fig. 5. Expression of E1, E1-L, and GmFTs. (A) Semiquantitative RT-PCRanalysis of E1 and E1-L expression levels in different tissues of Harosoy-E1plant under long-day conditions (18h light/6 h dark). All tissues were sam-pled on day 90 after emergence except for young roots and cotyledons onday 15. Images shown are representative profiles from three independentexperiments. E1-L, E1 paralogues (Glyma04g24640.1/Gm18g22670); GmTu-bulin, internal control. (B) Semiquantitative RT-PCR analysis of E1, E1-L,

GmFT2a, GmFT5a, and GmLCL2 expression levels in the leaves on day 15after emergence under 12 h light/12 h dark (short day; SD) and 16 h light/8 hdark (long day; LD) conditions. Images shown are representative profilesfrom three independent experiments. GmFT2a, Glyma16g26660.1; GmFT5a,Glyma16g04830.1; GmLCL2, G. max LHY/CCA1Like2 gene (EU076434);GmTubulin, internal control. Black and white bars represent dark and lightperiods, respectively. (C) Real-time quantitative RT-PCR analysis of E1,GmFT2a, and GmFT5a expression levels. Relative expression levels toGmTubulin (TUB) are shown. Values represent mean ± SD (n = 3) from threebiologically independent leaf samples from different plants. The experimentwas repeated once, and similar results were obtained both times. Black andwhite bars represent dark and light periods, respectively. H_E1, Harosoy-E1;H_e1, Harosoy (e1). (D and E) Semiquantitative RT-PCR analysis of E1 (e1)and GmFTs expression levels in leaves on day 15 after emergence under anintermediate light regime (14.5 h light/9.5 h dark). Images shown are rep-resentative profile from three independent experiments. The genotypes atthe E1, E3, and E4 loci are indicated at the bottom. The cultivars are Harosoy-E1, Harosoy (e1), NILs for the E1 locus [1-136(E1) and 1–136(e1)], and NILs forthe E4 locus (130E and 130L; Table S2).

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These results demonstrated that independent mutations in keygenes could explain a large proportion of the phenotypic varia-tion in rice. Likewise, the allelic variation in E1 was clearly re-lated to the flowering time phenotype in the present study. If E1is considered the fully functional WT allele, the e1-as allelerepresents a partially functioning allele and the e1-fs and e1-nlalleles are probably nonfunctional. The early-flowering Swedishcultivar Fiskeby V carries e1-nl, whereas Sakamotowase (e1-fs)and Toyosuzu (e1-nl) were developed in Hokkaido, Japan’snorthern island. Similarly, the Hejian 1 and Heihe 28 cultivars(e1-nl) were bred in Heilongjiang, a northern province of China.At these high latitudes, photoperiod insensitivity is generallya prerequisite for successful cultivation of a soybean cultivar.Various mutations of E1 might therefore have been preserved byhuman selection for cultivars adapted to a local photoperiodregime. Further genomic association study of the allelic varia-tions at E1 and other E loci among a large number of soybeancultivars and natural accessions of the wild species Glycine sojafrom different geographic locations will help us to understandthe domestication process.Flowering-time genes are generally considered to have pleio-

tropic effects on important traits such as yield, plant height, andstress tolerance. QTLs, such as those associated with chillingresponse (68), seed yield, and chemical composition (http://www.soybase.org), are tightly linked to the E1 locus. Further charac-terization will reveal whether these influences are from E1 itselfor from independent neighboring genes.

Putative Function of E1 in Photoperiodic Flowering of Soybean. Lightsignals are mainly perceived and mediated by several photo-receptors, including phytochromes and cryptochromes, mainly inthe leaves (69). A strong influence of phytochrome A on flow-ering signaling was observed in pea (Pisum sativum), a long-dayplant (70), and in rice, a short-day plant (71). In soybean, bothGmPHYA2 (E4) and GmPHYA3 (E3) suppress GmFTs(GmFT2a andGmFT5a) independently or jointly under long-dayconditions (38). Moreover, comparison of the transcriptionalprofiles in soybean seedlings between the E1 NILs showed thatthe E1 locus suppresses flowering by mediating the expressionlevels of GmFTs (40). In the present study, the lower expressionof E1 in Kariyutaka and other NILs with loss-of-function allelesofGmPHYA (e3 and e4) was coupled with an elevated expressionof GmFT2a or GmFT5a. Research on soybean plants undervarious photoperiod and light quality conditions showed that E1,E3, and E4 are all involved in photoperiod responses (20, 30).Allelic variations at the E loci, especially at E1, E3, and E4,

could therefore provide considerable genetic plasticity that wouldallow soybean to be cultivated at a range of latitudes (30). Ina mapping population with an e3 background, photoperiod in-sensitivity was observed in the e1E4, E1e4, and e1e4 genotypes,indicating that GmPHYA2 (E4) and E1 might concurrently me-diate photoperiodic flowering, at least partially, via a sharedpathway (20). Similarly, in the E3E4 or e3E4 genetic background,a major QTL was detected at the E1 locus (41). When the red tofar-red ratio was changed from 2.0 to 1.0, strong responses tolong-day conditions for both GmPHYA loci (E3, E4) and thestrongest response for the E1 locus were observed (30). The highsensitivity of E1 to light quality might result from convergence ofdifferent signaling pathways regulated by GmPHYA2 (E4) andGmPHYA3 (E3), and potentially by other photoreceptor genes.The expression ofE1was induced under long-day conditions, withno significant difference between the E1 and e1-as alleles. Inaddition, high-level expression of E1 was inversely related toGmFT expression. Therefore, we propose that E1 is a part of the

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Fig. 6. High expression of E1 in transgenic lines leads to late flowering. (A)Semiquantitative RT-PCR analysis of E1, E1-L and GmFT expression levels intransgenic plants as a function of the copy number of the E1 transgene.Images shown are representative profiles from three technical replicates asa result of the limitation of plant materials. T1 plants TG2-1 to TG2-5 werederived from the T0 transgenic plant TG2, whereas T1 plants TG4-1 and TG11-1 were derived from TG4 (T0) and TG11 (T0), respectively (Table S3). VC,transformation vector only (i.e., vector control); WT, Kariyutaka; R1, daysfrom emergence to opening of the first flower. (B) Real-time quantitativeRT-PCR analysis of E1, GmFT2a, GmFT5a, and E1-L expression levels in thetransgenic plants using the same biological samples as A. cDNA samples fromthe cultivar Clark under the same growth conditions were used as a control,whose expression level was set to 1 for all genes analyzed to linearly adjustthe corresponding gene expression levels in transgenic plants. Values rep-

resent means (n = 3), except for TG2-1 (n = 1). (C) No flower had beenproduced by day 30 in a transgenic plant (TG2-3) with high E1 expression,whereas flowers emerged in the VC, the WT, and a transgenic plant (TG2-1)with low E1 expression.

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phytochrome A signaling pathway and controls two functionallycoordinatedGmFT genes (GmFT2a andGmFT5a; Fig. 7). In thisscenario, long-day conditions are necessary for the induction ofE1 expression, whereas loss-of-function alleles at E3 or E4 canresult in some degree of suppression of E1 transcription andcorrespondingly elevated GmFT expression, leading to relativelyearly flowering.In the present study, we proposed a gene network for the reg-

ulation of flowering in soybean, but more work will be necessary tointegrate other factors into this network [e.g., to account for thefunction of the cryptochrome (i.e., GmCRY), a flavin-containingblue light photoreceptor in flowering]. GmCRY1a proteinshowed a photoperiod-dependent rhythmic expression patternthat was correlated with photoperiodic flowering and the lat-itudinal distribution cline of soybean cultivars (72).To further understand the function of E1, we will need to

identify the other genes and factors involved in the E1-mediatedphotoperiodic flowering pathway, and to further investigate howthese components control recognition of the critical day lengthfor flowering in soybean. Successfully deciphering of the E1gene will lead to a greater understanding of the exquisite co-ordination within soybean’s photoperiodic flowering-gene net-work, possibly leading to the establishment of a model forrelated plant species.

Materials and MethodsPlant Materials and Phenotypic Flowering Time Parameters. Cultivars wereobtained from the Japanese National Institute of Agrobiological SciencesGenebank and from the US Department of Agriculture Agricultural ResearchService National Plant Germplasm System (Table S2). The phenotypic pa-rameter flowering time (R1) was defined as the time from emergence toopening of the first flower (43).

Fine Mapping of E1 Locus. We crossed two NILs, Harosoy-E1 (L68-694,E1e2E3E4e5, PI547707) and Harosoy (e1) (L58-266, e1e2E3E4e5, PI547676), in2004. We confirmed the production of true F1 hybrid seed by using het-erozygous alleles in the E1 region based on the Satt365 and Satt557 markers.The F2 plants (117 plants) in 2005 and an F2:3 population of 1,442 individualsin 2006 were planted at Matsudo, Japan (35°78′N, 139°90′E). From 2007 to2009, all field trials were performed at National Institute of AgrobiologicalSciences (36°02′N, 140°11′E) at Tsukuba, Japan. We genotyped all progeniesby using the derived cleaved amplified polymorphic sequence marker TI (Fig.

S4), which can distinguish E1 from e1-as, to determine whether allelic vari-ation in E1 was correlated with phenotypic differences in 2009 (Fig. S1).

BAC Contig Construction and Marker Development. Twoparallel contigs (Fig. S2)for dominant and recessive E1genotypesweredeveloped from theMisuzudaizu(E1) BAC library (73) and the Williams 82 (e1-as) GM_WBb BAC library (https://www.genome.clemson.edu) using locus-specific markers (Table S4). BACscreening and BAC clone information are given in SI Materials and Methods.

Simple-sequence-repeat marker information was retrieved from SoyBase(http://www.soybase.org) and elsewhere (74, 75). Amplified fragment lengthpolymorphism markers were generated following a standard protocol (75).All markers (Table S4) used for fine-scale mapping of the E1 locus weredeveloped by targeting polymorphisms between the contigs (Fig. S2) rep-resentative of the two E1 genotypes.

Sequence Alignment and Phylogenetic Analysis. Sequences of E1 and itshomologues were aligned by using Clustal X2, and phylogenetic analysis wasperformed by using MEGA4. Sequence acquisitions and parameters used inClustal X2 and MEGA4 are provided in SI Materials and Methods.

Sequence Variation at E1 Locus in Different Cultivars. We amplified genomicregions (∼4 kb) including the promoter and coding regions of E1 from Harosoy-E1, Harosoy (e1), Sakamotowase,Williams 82,Misuzudaizu, andMashidougong503 by using the 4K primer (Table S4). These sequences were deposited in DNAData Base in Japan under accession numbers AB552966 to AB552971.

Screening and Characterization of EMS-Derived Mutations. Details of muta-genesis of OLERICHI50 and Fukuyutaka, Targeting Induced Local Lesions InGenomes screening of the mutant libraries, and phenotypic characterizationof the mutants are given in SI Materials and Methods.

Transformation of E1 into Kariyutaka. We amplified a 4,332-bp fragment(AB552966) from Harosoy-E1 using the 4K primer pair (Table S4), cloned itinto the pMDC123-GFP vector (58), and transformed this construct intoKariyutaka. The transformation followed a previously described procedure(58). The flowering times of the transgenic plants were evaluated in agrowth chamber with cool white fluorescent light plus incandescent lamps(235–270 μmol·s−1·m−2) under a 16 h light/8 h dark photoperiod at a constanttemperature of 25 °C.

RNA Preparation and Quantitative PCR. Plants were grown in growth cham-bers. RNA was extracted by using the TRIzol (Life Technologies) method.cDNA synthesis was performed by using ReverTra Ace (Toyobo).Semiquantitative RT-PCRs. PCR products were resolved in nondenaturingpolyacrylamide gel and fluorescently stained.Real-time quantitative RT-PCR. Transcript levels were quantified by using real-time quantitative RT-PCR (iCycle iQ; BioRad). Details are given in SI Materialsand Methods.

Subcellular Localization. To obtain a C terminus fusion plasmid, we amplifiedacDNA fragment containing the coding regionwithout a stop codonbymeansof PCRusing the primers 5′-CCATCGATAGATGAGCAACCCTTCAGATGAAAGG-3′ (forward) and 5′-GACTAGTATAATTCTCTGGCATAGCTTGTTTAAGG-3′ (re-verse) from plasmids (pGEM-T Easy; Promega) containing the E1, e1-as, or e1-fs sequence. The PCR product was then inserted downstream of the CaMV 35Spromoter and in-framewith the 5′ terminus of the eGFP gene in the backboneof the pBSK vector (76). The recombinant fusion plasmids were introducedinto onion epidermal cells by means of particle bombardment, and wereexpressed in Arabidopsis protoplasts by means of polyethylene glycol-medi-ated transfection, as described previously (77, 78).

ACKNOWLEDGMENTS.We thank Profs. S. Y. Chen andW. K. Zhang (Instituteof Genetics and Developmental Biology, Chinese Academy of Sciences) forinstruction in and generous help with functional analysis; N. Jiang (MichiganState University), E. R. Cober (Agriculture and Agri-Food Canada), J. D. Faris(US Department of Agriculture Agricultural Research Service), L. Yan(Oklahoma State University), T. Izawa (National Institute of AgrobiologicalSciences), and B. Larkins (University of Arizona) for critical reading of anearly draft of this paper; K. Saeki (Nara Women’s University) for interpreta-tion of the results; and J. Abe (Hokkaido University) and B. Liu and F. Kong(Northeast Institute of Geography and Agroecology, Chinese Academy ofSciences) for providing primer information. This study was supported byMinistry of Agriculture, Forestry and Fisheries of Japan Genomics for Agri-cultural Innovation Grants DD-2040 and SOY2003; Japan Grant-in-Aid forScientific Research (A) 18208001; and Chinese Academy of Sciences HundredTalents Program and Grant KZCX2-EW-303.

E3 E4GmFT2a/5a GmFT2a/5a

G FTs

unknown factors

G FTs

Leaves Leaves

ApexApex

XE1E1 SD LD

LightEarly flowering

Late flowering

?

Fig. 7. A proposed flowering-time gene network in soybean. Transcriptionof E1 antagonistically determines the expression level of GmFTs (GmFT2aand GmFT5a), thereby controlling photoperiodic flowering. Arrows repre-sent stimulation of gene expression; T-shaped symbols represent inhibitionof gene expression; X represents the negation of inhibition/promotion; SD,short day length; LD, long day length.

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