RESEARCH ARTICLES

Ln Is a Key Regulator of Leaflet Shape and Number of Seedsper Pod in SoybeanW

Namhee Jeong,a Su Jeoung Suh,a Min-Hee Kim,a Seukki Lee,b Jung-Kyung Moon,b Hong Sig Kim,c

and Soon-Chun Jeonga,1

a Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology, Cheongwon, Chungbuk 363-883, Republic of KoreabNational Institute of Crop Science, Rural Development Administration, Suwon 441-857, Republic of KoreacDepartment of Crop Science, Chungbuk National University, Cheongju 361-763, Republic of Korea

Narrow leaflet soybean (Glycine max) varieties tend to have more seeds per pod than broad leaflet varieties. Narrow leaflet insoybean is conferred by a single recessive gene, ln. Here, we show that the transition from broad (Ln) to narrow leaflet (ln) isassociated with an amino acid substitution in the EAR motif encoded by a gene (designated Gm-JAGGED1) homologous toArabidopsis JAGGED (JAG) that regulates lateral organ development and the variant exerts a pleiotropic effect on fruitpatterning. The genomic region that regulates both the traits was mapped to a 12.6-kb region containing only one gene, Gm-JAG1. Introducing the Gm-JAG1 allele into a loss-of-function Arabidopsis jagged mutant partially restored the wild-type JAGphenotypes, including leaf shape, flower opening, and fruit shape, but the Gm-jag1 (ln) and EAR-deleted Gm-JAG1 alleles in thejagged mutant did not result in an apparent phenotypic change. These observations indicate that despite some degree offunctional change of Gm-JAG1 due to the divergence from Arabidopsis JAG, Gm-JAG1 complemented the functions of JAG inArabidopsis thaliana. However, the Gm-JAG1 homoeolog, Gm-JAG2, appears to be sub- or neofunctionalized, as revealed by thedifferential expression of the two genes in multiple plant tissues, a complementation test, and an allelic analysis at both loci.

INTRODUCTION

Leaves and flowers develop continuously at the flanks of theshoot apical meristem in flowering plants. A single mutationoften causes pleiotropic phenotypes during leaf and flower de-velopment (Tsukaya, 2006), suggesting that a common regula-tory circuit is involved in the production of leaves and flowers. Forexample, Leafy/UNIFLOLIATA regulates both leaf and flower mor-phogenesis in pea (Pisum sativum; Hofer et al., 1997). Conversely,combinations of floral homeotic mutations result in the conversionof floral organs to leaf-like structures (Bowman et al., 1993). Thepea mutant crispa (cri ), which is defective in PHANTASTICA(PHAN), has a reduced leaflet width-to-length ratio and exhibitspleiotropic effects, including longer internodes, reduced pedunclelength, and lower seed production per pod (Tattersall et al., 2005).However, the molecular genetic basis of the relationship betweenleaf/flower development and pods or fruits, which are derivedfrom the flower and associated tissues, is poorly understood.

Seed yield is determined by the number of seeds per unit areaand seed weight. During soybean (Glycine max) production, thenumber of seeds per unit area is a product of the number of plantsper unit area, the number of pods per plant, and the number ofseeds per pod (NSPP) (Pedersen and Lauer, 2004). The NSPP is

primarily determined by the number of ovules per placenta aswell as the frequency of embryonic abortions. Soybean ovariescontain from one to four ovules, indicating that the NSPP isdetermined at the early stage of flower development (Carlsonand Lersten, 2004). A quantitative trait loci (QTL) analysis usingthree recombinant inbred populations derived from reciprocalcrosses of three cultivars demonstrated that the average NSPPin soybean is determined genetically by multiple significant QTLthat account for ;50% of the heritable variation (Tischner et al.,2003). These QTL were linked to the Ms1, Ms6, or St5 genes formale and female sterility and Lf1 and ln for leaflet number andleaflet shape, respectively, although further molecular geneticanalyses have not been conducted. Among the genes associ-ated with NSPP, the ln gene has been more frequently studied,as the ln locus named after narrow leaflet has long been sug-gested to be tightly linked to the NSPP trait or to exert a majorpleiotropic effect on the NSPP trait (Takahashi, 1934; Domingo,1945; Bernard and Weiss, 1973). However, the genetic relation-ship between the two traits has not been easy to resolve partlydue to additional minor modifying gene(s) in the ln genetic back-ground (Weiss, 1970; Jeong et al., 2011).Previous agronomic studies on the ln locus using isogenic

lines (Mandl and Buss, 1981), diverse cultivars (You et al., 1995),and broad (Ln/Ln), heterozygous (Ln/ln), and narrow (ln/ln) leaflettypes (Dinkins et al., 2002) have repeatedly found that broad andnarrow leaflet soybean genotypes have similar seed yields, butnarrow leaflet plants consistently produce smaller seeds thanthose of broad leaflet plants. The fact that the narrow leaflet plantstend to produce a greater number of seeds has garnered a greatdeal of attention, particularly in the development of soybean cultivars

1 Address correspondence to scjeong@kribb.re.kr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Soon-Chun Jeong(scjeong@kribb.re.kr).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.104968

The Plant Cell, Vol. 24: 4807–4818, December 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.

for sprouts. The number of seeds is a critical yield componentfor soybean sprout production (Lee et al., 2001), and this hasresulted in a narrow leaflet shape in most of the sprout soybeancultivars recently developed in Korea (Yu et al., 2008). Collec-tively, the causes of this association might include either thetight linkage of genes that control independent traits or thepleiotropic effects of the target gene for both traits.

Cloning and functional understanding of loci regulating theyield components may provide molecular genetic tools for im-proving yield, one of the most complex plant traits. In this study,we performed map-based cloning of the ln locus, a regulator ofleaflet shape and NSPP. Our results indicated that the leaflet-shape and NSPP traits are pleiotropic effects of the ln gene.Consequently, our results should facilitate development of newsoybean cultivars with high yield potential.

RESULTS

Physical Map

Our fine genetic map was used to delimit the genomic regionthat regulates both leaflet shape and NSPP traits between mi-crosatellite markers Ln_at004 and Ln_atre04 on soybean chro-mosome 20 (Jeong et al., 2011), a region that has a sequencelength of 66 kb in the soybean genome (Schmutz et al., 2010),corresponding to 0.7 centimorgans (Figure 1A). To further dis-sect the 66-kb genomic region, 4219 F3 seedlings derived from162 F2 plants heterozygous for both Ln_at004 and Ln_atre04were grown and screened for recombination between Ln_at004and Ln_atre04. As a result, 17 recombinants with the genotypehomozygous at one marker and heterozygous at the other wereselected. The recombinants and their progeny were grown fora phenotypic evaluation of leaflet shape and NSPP traits. By furthergenotyping the recombinant plants using markers Ln_43k, Ln_44k,Ln_attre, Ln_AH, Ln_54k, and Ln_57k (see Supplemental Table 1online), the ln locus was localized to the interval between Ln_44k andLn_57k (Figure 1B). Three markers (Ln_attre, Ln_AH, and Ln_54k)cosegregated with ln. One recombination event between Ln_44kand ln and one recombination event between Ln_57k and ln weredetected. Thus, the mutation responsible for the derivation of bothnarrow leaflet shape and high NSPP value in soybean was delimitedto a 12.6-kb region between Ln_44k and Ln_57k (Figure 1C).

Only one gene (Glyma20g25000.1) has been predicted in this12.6-kb region of the Glyma1.0 soybean gene annotation da-tabase (accessible at Phytozome v5.0, www.phytozome.net,accessed August 2012) (Figures 1C and 1D). When we con-ducted de novo gene annotation with AUGUSTUS (Stanke et al.,2006) for this sequence and queried Next-Gen Sequence Data-bases for small RNAs (http://mpss.udel.edu/soy_sbs/, accessedAugust 2012), no additional gene or small RNA could be pre-dicted. The gene was predicted to be homologous to Arabidopsisthaliana JAGGED (JAG), which was reported to be involved inthe development of lateral organs, including leaves and flowers(Dinneny et al., 2004; Ohno et al., 2004), and its coding region wasphysically located between 34,688,514 and 34,690,379 bp on theGm20 pseudomolecule of soybean chromosome 20 (http://www.phytozome.net). A BLAST search against the soybean genomesequence revealed that Glyma20g25000.1 has a close paralog

gene, Glyma10g42020.1, on the Gm10 of soybean chromosome10. Glyma20g25000.1 and Glyma10g42020.1 were named Gm-JAG1 and Gm-JAG2, respectively.BLAST searches of the 12.6-kb region against the GenBank

EST database hit more than 10 ESTs, including GD767462 andGD866768, corresponding to the 59 untranslated and 39 un-translated regions of the annotated gene, respectively. However,alignment of those ESTs did not reveal a full-length open readingframe (ORF) and suggested different coding sequences from theannotated Glyma20g25000.1 and Glyma10g42020.1. Thus, theORF of Gm-JAG1 was determined by cloning and then by se-quencing RT-PCR products from RNA isolated from the shoottips of ‘Sowon’ (ln) and ‘V94-5152’ (Ln) using the primer pairsdesigned from the exon regions flanking the included intron (seeSupplemental Table 2 online). Most of the RT-PCR productscontained sequences of both Gm-JAG1 and Gm-JAG2, exceptthe product of a cJAG-m-3 primer pair. Thus, the ORF sequenceof Gm-JAG2 was also completely determined using an additionalcJAG-mh-3 primer pair. A comparison of the multiple RT-PCRproduct sequences revealed that although rare, Gm-JAG1 con-tains an alternative splicing site of 6 bp (two amino acids) on the59 side of the third exon (Figure 1D). Thus, Gm-JAG1 encodes 256(predominant) or 258 amino acid proteins. Gm-JAG1 and Gm-JAG2 showed 90.7% identity at the amino acid level of theirpredicted coding regions (see Supplemental Figure 1 online).Four conserved domains were readily identified in the pre-

dicted Gm-JAG1 and Gm-JAG2 sequences when comparedwith Arabidopsis JAG and its paralog NUBBIN (NUB), tomato(Solanum lycopersicum) JAG, and maize (Zea mays) JAG: anEAR motif, a putative nuclear localization signal sequence, asingle C2H2-type zinc finger motif, and a Pro-rich motif (Figure1E; see Supplemental Figure 1 online). The putative nuclear lo-calization signal sequence and Pro-rich motif are poorly con-served in NUB and Zm-JAG. Interestingly, our BLAST searchesindicated that no soybean homolog of the NUB gene is presentin the current soybean genome sequence assembly (Schmutzet al., 2010). A 1-bp substitution or change from Asp (Ln) to His(ln), which is the only difference between the ORF sequences ofthe Gm-JAG1 genes in V94-5152 (Ln) and Sowon (ln) plants,occurred at the EAR motif. As the conserved EAR motif con-tained a nonsynonymous mutation, it could be hypothesizedthat the mutation might be responsible for the phenotypic dif-ferences between the Ln and ln plants. Outside of these de-scribed domains, amino acid sequence conservation betweenGm-JAG1 and At-JAG/Sl-JAG was low, indicating possible func-tional dissimilarities (see Supplemental Figure 1 online).A comparison of the 12.6-kb sequences between the mapping

parents revealed eight polymorphisms between two single nu-cleotide substitutions for markers Ln_44k and Ln_57k (Figures 1Cand 1D). These included five polymorphisms in the putative pro-moter region within 1.5 kb upstream of the 59 untranslated exon,one in the 59 untranslated exon, one in the second exon, and one(Ln_54k site) 39 downstream of the stop codon.

The ln Phenotype and Its Expression

Differences in leaflet shape and pod type between Ln and ln soy-bean plants have been repeatedly assessed in different soybean

4808 The Plant Cell

Figure 1. Molecular Cloning of ln.

Control of Fruit Patterning by Ln 4809

genetic backgrounds (Takahashi, 1934; Domingo, 1945; Weiss,1970; Bernard and Weiss, 1973; Mandl and Buss, 1981; Youet al., 1995; Dinkins et al., 2002; Jeong et al., 2011). These studieshave consistently shown that Ln plants tend to have broaderleaflet shapes (Figures 2A and 2B) and lower NSPP than ln plants(Figures 2C and 2D). As the mutations of the Arabidopsis JAGgene, the soybean homolog of which is presumed to encode Lnby the physical mapping described above, were reported todisplay defects in floral morphology including narrow floral or-gans (Dinneny et al., 2004; Ohno et al., 2004), we examinedmorphology of flowers. The flowers of ln plants (Figure 2F) ap-pear smaller than those of Ln plants (Figure 2E) likely becausefloral organs, including sepals and petals of ln flowers, aresmaller or narrower than those of Ln flowers. When we mea-sured width of banner petals (Figure 2H) and length of carpels(Figure 2I), those of ln plants were found to be significantlynarrower and shorter than those of Ln plants (t test, P < 0.01). Anextra petal or carpel occasionally appears in the ln-containingparent Sowon (Figure 2G). However, these abnormal flowerslikely are not determined by ln, as they appear in both Ln- andln-recombinant plants.

To investigate whether promoter region mutations are re-sponsible for the soybean leaflet shape and NSPP, JAG1 tran-script levels in different soybean lateral organs of V94-5152 (Ln)and Sowon (ln) plants were examined using RT-PCR. The pat-terns of RNA accumulation in both Ln and ln plants were quitesimilar to each other. The JAG1 transcript was detected duringvegetative and reproductive development in the shoot apex(meristem) and in open flowers (Figure 2J), as observed for At-JAG in different lateral organs of Arabidopsis (Ohno et al., 2004).In soybean, a low level of RNA transcript was detected in youngln pods, but not in those of Ln at a higher cycle number. Vali-dation of these results with quantitative RT-PCR (qRT-PCR)analysis suggested that JAG1 is most strongly expressed in themeristem and flowers of both Ln and ln plants and is greatlyreduced or not detectable in other organs (Figure 2K). Despitethe lack of expression studies for this gene at the tissue levelconducted previously in Arabidopsis studies (Dinneny et al.,2004; Ohno et al., 2004), these results indicate that the mutationsin the promoter region of Gm-JAG1 did not affect overall mRNAexpression patterns of the gene. Furthermore, as we observedonly a single nucleotide substitution in the coding region of theGm-JAG1 gene, which led to a single amino acid change, oursubsequent analyses focused on determining whether this was

the causal mutation of the phenotypic differences between theLn and ln genotypes.

Complementation of the Arabidopsis jag-3 Mutantwith Gm-JAG1

To validate the function of Gm-JAG1 for broad leaflet and lowNSPP (versus Gm-jag1 for narrow leaflet and high NSPP), weintroduced the V94-5152 GmJAG1 allele driven by the Arabi-dopsis JAG promoter (PAtJAG:gGmJAG1) into an Arabidopsisjag loss-of-function mutant (jag-3). Since Sowon is not readilytransformed using current technology, complementation of theln phenotype with the Ln Gm-JAG1 allele was not performed.The jag-3 mutant expresses a defective truncated JAG proteinby disruption of the 39-splice acceptor sequence (Ohno et al.,2004). The presence of the soybean Gm-JAG1 allele in thetransgenic lines was detected by PCR analysis and sequencingof PCR fragments and then further verified by RT-PCR (Figure 3A).The transgene (Figures 3D and 3H) showed substantial rescue ofthe jag-3 phenotypes in 34 of 107 primary transformants. Notably,the transformation of Gm-JAG1 converted narrow leaves andfloral organs of the Arabidopsis jag-3 mutant into relatively broadleaves and floral organs, nearly the same characteristics as wild-type Arabidopsis Landsberg erecta (Ler) (Figures 3B to 3D and 3Fto 3H). The serrated leaves (fifth leaf) in the PAtJAG:gGmJAG1transgenics appeared slightly later than those in jag-3 (fourth leaf)but appeared slightly earlier than in the wild type (seventh leaf)(Figures 3F to 3H). However, extra petals and rosette leaves rel-ative to numbers in the wild type as previously reported for jag-3(Ohno et al., 2004) were occasionally observed in the pAtJAG:gGmJAG1 transgenics (Figures 3B to 3D and 3F to 3H), indicatingthat the transformation of Gm-JAG1 did not rescue these ab-normalities in a notable manner. These results suggest that, de-spite substantial divergence in amino acid sequence, Gm-JAGand Arabidopsis JAG show a high degree of functional homology.The question remained of whether the one nonsynonymous

nucleotide substitution (Gm-jag1 allele) detected at the EARmotif region of the Gm-JAG1 locus in the cultivated soybeanhad no or diminished functions relative to the Gm-JAG1 allele forleaflet shape and NSPP. To address this question, we introducedthe Sowon Gmjag1 allele driven by the Arabidopsis JAG promoter(PAtJAG:gGmjag1) into the jag-3 mutants and obtained 63 trans-genic (Gmjag1) lines. Expression of the Gmjag1 allele was con-firmed by RT-PCR (Figure 3A). All PAtJAG:gGmjag1 transgenics

Figure 1. (continued).

(A) Chromosomal location of ln determined by genetic linkage mapping on chromosome (Chr) 20.(B) Fine mapping of ln. Vertical lines indicate microsatellite and single nucleotide polymorphism markers. Markers are indicated above the line. Thenumber of recombinants between ln and each marker left in the chromosomal interval after evaluation of the marker is indicated below the line.(C) Sequence map between markers Ln_44k and Ln_57k. Mutations found between the mapping parents are labeled below the map: Insertion/deletionpolymorphisms are indicated by closed triangles and single nucleotide substitutions by open triangles.(D) JAG1 structure showing five exons (gray boxes), four introns (white boxes), and 1.5 kb of the putative promoter region. Start and stop codons(vertical lines) and a 6-bp alternative splicing site (arrow) of the gene are labeled.(E) Amino acid sequence of JAG1. Position and nature of the ln (asterisk) mutation is indicated. Two amino acids changed by an alternative splicing of 6bp between the second intron and third exon are boxed. The four conserved regions that are highlighted in bold and solid lines are indicated. NLS,putative nuclear localization signal sequence.

4810 The Plant Cell

showed phenotypes identical to that of the Arabidopsis jag-3mutant (Figures 3C, 3E, 3G, and 3I). These results substantiatedthat the mutation at the EAR motif is sufficient for the conversionof Gm-JAG1 to Gm-jag1.

Although JAG promotes the formation of the valve margin thatfacilitates the detachment of the valves from the replum duringdevelopment of Arabidopsis fruit, which is also referred to as si-lique (Dinneny et al., 2005), the role of JAG in silique growth andseed set has been poorly characterized. To substantiate commonfunctional homology between Arabidopsis JAG and Gm-JAG1 interms of the fruit development, it was necessary to examinechanges in silique morphology, seeding pattern in the siliques,

length of siliques, and the number of seeds per silique in theArabidopsis wild-type, jag-3, Gm-JAG transgenics, and Gm-jagtransgenics. The siliques in wild-type plants were longer andthicker than those in the jag-3 plant (Figures 3J and 3K). How-ever, seeds were more densely packed in the siliques in jag-3than those in the wild type (Figures 3N and 3O). The Gm-JAG1transgenic lines (Figures 3L and 3P) showed substantial rescueof the jag-3 silique phenotypes. However, the siliques of theGm-jag1 transgenic lines were similar in morphology to those ofjag-3 (Figures 3M and 3Q). Silique length was measured for eachgenotype, and seeds from one side of the septum after removinga valve in each silique were counted for each genotype. Silique

Figure 2. The ln Phenotype and Its Expression Pattern in Plant Tissues.

(A) and (B) Trifoliolate leaves of V94-5152 (Ln) and Sowon (ln). Bar = 50 mm.(C) and (D) Pod types occurring in Ln (C) and ln (D) allele plants. Predominant two-seeded pods are indicated by an asterisk. Bar = 10 mm.(E) and (F) Ln whole flower (E) and ln whole flower (F) and their detached banner petals, wing petals, keel petals, and carpels. Bar = 5 mm.(G) Abnormal flowers having two banner petals, three keel petals, and two carpels, respectively. Bar = 5 mm.(H) and (I) Distributions of banner petal width (H) and carpel length (I) in Ln and ln plants (n = 21). Perpendicular lines span from maximum to minimum ofdata, boxes span the interquartile range, and the horizontal lines indicate the median.(J) RT-PCR analysis of Gm-JAG1 in different tissues of ln and Ln allele plants. Numbers refer to PCR cycles. RNA was extracted from tissues of field-grown plants. Amplification of actin 11 was used as a control.(K) Quantitative RT-PCR analysis of Gm-JAG1 expression levels in different tissues of ln and Ln allele plants. Two biological replicates and threetechnical replicates were performed. Values were normalized to the expression of Actin 11 and are expressed relative to the level (set to 1.0) in themeristems of the Ln plants. Error bars indicate SE.

Control of Fruit Patterning by Ln 4811

length and number of seeds in siliques were significantly longerand higher, respectively, in the wild type than those in jag-3 inthe Fisher’s Protected LSD test (see Supplemental Figure 2online). The number of seeds per unit silique length was significantlylower in the wild type than in jag-3. Silique length and number ofseeds in the Gm-JAG1 transgenic lines were significantly shorterand lower than in the wild type but significantly longer and higherthan those in jag-3. Collectively, our results suggest that introducing

Gm-JAG1 into the jag-3 mutant partially rescued the wild-type si-lique phenotypes by restoring silique length and seed distribution.

Functional Divergence between Soybean JAG1and Arabidopsis JAG

Our genetic and physical maps and complementation tests in-dicated that the single nucleotide substitution at the EAR motif

Figure 3. Functional Analysis of Gm-JAG1 and Gm-jag1 Alleles in the Arabidopsis jag-3 Mutant.

(A) Confirmation of expression of soybean Gm-JAG1 and Gm-jag1 alleles in the transgenic Arabidopsis jag-3 lines by RT-PCR analysis. RNA wasextracted from inflorescence tissues of PAtJAG:gGmJAG1 transgenic plants, PAtJAG:gGmjag1 transgenic plants, wild-type Ler, and the jag-3 mutant.(B) to (E) Flower morphology. Flowers of Ler (B), jag-3 (C), PAtJAG:gGmJAG1 (D), and PAtJAG:gGmjag1 (E). Bar = 1 mm.(F) to (I) Leaf morphology. Rosette leaves of Ler (F), jag-3 (G), PAtJAG:gGmJAG1 (H), and PAtJAG:gGmjag1 (I). Leaves were excised from 4-week-oldplants. The first leaves are shown at left. Bar = 10 mm.(J) to (Q) Fruit morphology. Fully elongated fruits of Ler (J), jag-3 (K), PAtJAG:gGmJAG1 (L), and PAtJAG:gGmjag1 (M). Bar = 1 mm.(N) to (Q) Morphology of dehiscent fruit. Dehiscent fruits of Ler (N), jag-3 (O), PAtJAG:gGmJAG1 (P), and PAtJAG:gGmjag1 (Q). A valve was removedfrom each dehiscent silique to display the seeding pattern. Bar = 1 mm.

4812 The Plant Cell

of Gm-JAG1 was likely the causal mutation responsible for thephenotypic difference between Ln and ln soybean plants. How-ever, more mutations were observed at the Gm-JAG1 promoterregion and the sequence conservation between the ArabidopsisJAG and soybean JAG1 proteins was low outside of the con-served motifs, making it crucial to determine the extent to whichthe two proteins and their promoters are functionally similar. Toaddress this question, we introduced several different geneconstructs into the Arabidopsis jag-3 mutant. First, we com-pared the effect of Gm-JAG1 and Gm-jag1 misexpression in thejag-3 genetic background with the published effects of Arabi-dopsis JAG overexpression (Dinneny et al., 2004; Ohno et al.,2004) (Figure 4; see Supplemental Figure 3 online). The 35S:GmJAG1 transgenics displayed phenotypes highly reminiscentof PAtJAG:gGmJAG1 transgenic plants but did not display theaberrant phenotypes, including fusion of rosette leaves andstipule outgrowth from the lateral margins at the base of the leaf,observed in JAG overexpressing Arabidopsis. The 35S:Gmjag1transgenics showed phenotypes nearly identical to those of theArabidopsis jag-3mutant. These results demonstrate that although

Arabidopsis JAG and soybean JAG1 maintain common essen-tial functions observed in the initial lateral organ development,the substantial divergence in amino acid sequence mighthave led to diminished function of G. max JAG1 in ectopic tis-sues. Second, to investigate whether the promoter regions thathad more variation than coding regions in the comparison ofGm-JAG1 and Gm-jag1 sequences maintained any functionalconservation between soybean and Arabidopsis, V94-5152 Gm-JAG1 (PGmJAG1:gGmJAG1) and Sowon Gm-jag1 (PGmjag1:gGmjag1) driven by their own promoter, respectively, were in-troduced into the Arabidopsis jag-3 mutant. We obtained 20PGmJAG1:gGmJAG1 and 57 PGmjag1:gGmjag1 transgenics. RT-PCR did not detect expression of Gm-JAG1 and Gm-jag1 mRNA(Figure 4A), and all transgenics showed phenotypes identical tothat of the Arabidopsis jag-3 mutant (see Supplemental Figure 3online). The results indicate that the promoter region of Gm-JAG1was no longer functional in Arabidopsis due to substantial di-vergence. Third, an EAR-deleted version of Gm-JAG1 (PAtJAG:gGmJAG1DEAR) was transformed into the Arabidopsis jag-3 mu-tant. We obtained 49 PAtJAG:gGmJAG1DEAR transgenic lines. All

Figure 4. Functional Analysis of 35S:GmJAG1, 35S:Gmjag1, PGmJAG1:gGmJAG1, PGmjag1:gGmjag1, PAtJAG:gGmJAG1DEAR, and PAtJAG:gGmJAG2 Gene Constructs in the Arabidopsis jag-3 Mutant.

(A) Confirmation of expression of the introduced gene constructs in the transgenic Arabidopsis jag-3 lines by RT-PCR analysis. RNA was extracted frominflorescence tissues of 35S:GmJAG1, 35S:Gmjag1, PGmJAG1:gGmJAG1, PGmjag1:gGmjag1, and PAtJAG:gGmJAG1DEAR transgenic plants, wild-type Ler, and the jag-3 mutant.(B) Confirmation of expression of the introduced gene constructs in the transgenic Arabidopsis jag-3 lines by RT-PCR analysis. RNA was extracted frominflorescence tissues of PAtJAG:gGmJAG2 transgenic plants, wild-type Ler, and the jag-3 mutant.(C) to (H) Fruit morphology. Fully elongated fruits of Ler (C), jag-3 (D), 35S:GmJAG1 (E), 35S:Gmjag1 (F), PAtJAG:gGmJAG1DEAR (G), and PAtJAG:gGmJAG2 (H). Bar = 1 mm.

Control of Fruit Patterning by Ln 4813

PAtJAG:gGmJAG1DEAR transgenics showed phenotypes nearlyidentical to those of the Arabidopsis jag-3 mutant and PAtJAG:gGmjag1 transgenic lines (Figure 4G; see Supplemental Figure 3online). The results substantiated that the mutation at the EARmotifis sufficient for the conversion of Gm-JAG1 to Gm-jag1 and alsoindicated that Gm-JAG1 without the EAR motif is nonfunctional.

Evolutionary Diversification of JAG in Soybean

The BLAST search indicated that Gm-JAG1 and its homoeologGm-JAG2 are located on large duplicated blocks betweensoybean chromosomes 20 and 10, respectively, in the soybeangenome, which were presumably generated by a soybean line-age–specific paleoallotetraploidy event dated to ;13 millionyears ago (Schmutz et al., 2010). Therefore, these two genes area homoeologous pair. The presence of only two copies of theGm-JAG homolog was substantiated by DNA gel blot analysis(see Supplemental Figure 4 online). The sequences of the Gm-JAG2 promoter and coding region, determined from Sowonand V94-5152, were identical, indicating that Gm-JAG2 is notresponsible for the phenotypic difference between Sowon andV94-5152. However, as amino acid sequences of Gm-JAG1 andGm-JAG2 are identical in the three conserved domains except thePro-rich domain out of the four conserved domains, Gm-JAG2may be functional. To address this question, we introduced Gm-

JAG2 driven by the Arabidopsis JAG promoter (PAtJAG:gGmJAG2)into the Arabidopsis jag-3 mutant. Surprisingly, 13 of 35 PAtJAG:gGmJAG2 transgenics showed phenotypes nearly identical toPAtJAG:gGmJAG1 transgenic plants relative to the wild-typeArabidopsis (Figure 4H; see Supplemental Figure 3 online).The expression patterns of Gm-JAG1 and Gm-JAG2 were

compared to better understand their functional diversity. RT-PCR and qRT-PCR were used to profile Gm-JAG2 expression indifferent lateral organs. Consistent with the expression patternsof Arabidopsis JAG, Gm-JAG1 was mainly expressed in themeristem and flowers (Figure 2J). By contrast, the Gm-JAG2transcript was strongly expressed in broader types of tissues thanwas Gm-JAG1 (Figure 5). At similar levels to that of the shootapex and open flowers, the Gm-JAG2 transcript was expressed inyoung pod tissues of both ln and Ln plants, and it was markedlydetected in young leaf tissues of Ln plants but greatly reduced inthose of ln plants (Figure 5B). These results indicate that Gm-JAG2 may be sub- or neofunctionalized.Allelic variation of Gm-JAG1 in the wild and cultivated soy-

bean populations was evaluated in the 59 accessions con-taining a broad leaflet type and in 12 accessions containinga narrow leaflet type. The genotypes scored by the Ln_AHmarker generated from the 1-bp substitution mutation at theEAR correlated perfectly with the genotypes scored by leaflettypes (see Supplemental Table 3 online). We observed no ad-ditional allelic variation presumed to affect the amino acid se-quence encoded by Gm-JAG1 from a comparison of sequencesof Gm-JAG1 determined from 14 accessions selected from theabove 71 accessions (see Supplemental Figures 5 and 6 online).

DISCUSSION

We isolated the classical soybean locus (ln) that shapes narrowleaflets and increases seed number in soybean by positionalcloning. Ln encodes Gm-JAG1, a homolog of an Arabidopsistranscription factor that acts to promote lateral outgrowth inaerial organs (Dinneny et al., 2004; Ohno et al., 2004). The geneexpression analysis indicated that the transition from dominantLn (V94-5152 cultivar) to recessive ln (Sowon cultivar) pheno-types in soybean was associated with a single nucleotide sub-stitution at a region encoding the Gm-JAG1 EAR motif, whichled to a single amino acid change. When Gm-JAG1 was in-troduced into the Arabidopsis jag-3 mutant, it restored the wild-type phenotypes of the flowers, leaves, and silique shape tosome degree. When Gm-jag1was introduced into the Arabidopsisjag-3 mutant, it did not restore the wild-type phenotypes, indi-cating that the single nucleotide mutation is equivalent to a loss-of-function mutation. Collectively, our results show that phenotypicvariations for narrow leaflet and high NSPP are predominantly theresult of the pleiotropic effects of the ln gene and thus provided ananswer to a long debated hypothesis of whether leaflet shapeand NSPP are regulated by the ln gene alone or by the tightlylinked loci including ln.The molecular basis of the relationship between floral organs

and leaves has been actively studied ever since the formation ofthe ABC model (Coen and Meyerowitz, 1991). While there aredistinct morphological differences in flower, leaf, and silique (fruit)

Figure 5. Expression Analysis of Gm-JAG2 in Different Tissues of ln andLn Allele Plants.

(A) RT-PCR analysis. Numbers refer to PCR cycles. RNA was extractedfrom tissues of field-grown plants. Amplification of actin 11 was used asa control.(B) qRT-PCR analysis of Gm-JAG2 expression levels in different tissuesof ln and Ln allele plants. Two biological replicates and three technicalreplicates were performed. Values were normalized to the expression ofActin 11 and are expressed relative to the level (set to 1.0) in the mer-istems of the Ln plants. Error bars indicate SE.

4814 The Plant Cell

shapes between the wild-type Arabidopsis and jag mutants andbetween the wild-type tomato and lyrate mutant, Ln (Gm-JAG1)and ln (Gm-jag1) soybean plants showed less distinct mor-phological differences in leaf, pod, and flower shapes. This mayhave been due to the extent of the mutation or differences in theorganization of development in soybean, Arabidopsis, or tomatoor due to the existence of a neo- or subfunctionalized homoe-ologous gene, JAG2. Similar observations have been reportedby functional studies of PHAN. phan mutations have similarphenotypes in tomato (Kim et al., 2003) and tobacco (Nicotianatabacum) (McHale and Koning, 2004), but in the homologousmaize rs2 and Arabidopsis as1 mutants, the leaves develop withessentially normal polarity (Timmermans et al., 1999; Tsiantis et al.,1999; Byrne et al., 2000). Unlike the cri mutant, another PHAN-defective pea mutant, which shows decreased seed production perpod in the mutant compared with the wild type (Tattersall et al.,2005), the ln mutant led to an increase in the NSPP.

The only difference between Sowon (ln) and V94-5152 (Ln) atthe coding region of Gm-JAG1 was a single nucleotide sub-stitution at the EAR motif region. The results from the expressionpatterns, allele diversity analysis, and transformations of theGm-jag1 and EAR-deleted Gm-JAG1 genes into the Arabidopsisjag-3mutant supported that the mutation was solely responsiblefor the phenotypic difference between Ln and ln. The EAR motifis found in broad gene families and is conserved across diverseplant species (Kagale et al., 2010). The EAR motif is composedof LxLxL, DLNxxP, or an overlap of the two (Kagale et al., 2010).The Gm-JAG1 EAR motif is composed of LDLNNLP. Gm-jag1contains LHLNNLP, in which the second D, Asp, which has anacidic side chain, is replaced by H, His, which has a basic sidechain. Arabidopsis JAG and tomato JAG contain LDLNNLP,supporting that the JAG EAR motif is conserved among differentplant species. The EAR motif contains alternating hydrophilicand hydrophobic amino acids, and the amphiphilic Asp is nec-essary for its repressive function (Ohta et al., 2001; Hiratsu et al.,2003). When the EAR motif is fused to transcription factors, thetranscription factors become repressors, and transcription fac-tors that activate other transcription factors become trans-repressors (Ohta et al., 2001). Thus, our finding that the singlemutation at the EAR motif leads to loss of JAG activity indicatesthat JAG functions as a repressor, thereby providing further in-sight into efforts to find the position of JAG in genetic regulatorynetworks that coordinate lateral organ development. Many genesupstream or downstream of JAG have been reported recently. JAGexpression is negatively regulated by the BLADE-ON-PETIOLEgenes in lateral organs, particularly in the Arabidopsis crypticbract (Hepworth et al., 2005; Norberg et al., 2005). JAG activityis negatively regulated by REPLUMLESS (Dinneny et al., 2005),which promotes replum development in Arabidopsis fruit (Roederet al., 2003). JAG, together with ASYMMETRIC LEAVES1 (AS1) andAS2, represses the expression of the boundary-specifying genesCUP-SHAPED COTYLEDONS1 and 2 and PETAL LOSS, whichmaintains the low cell proliferation rate near the organ primordiafor the development of the sepal and petal (Xu et al., 2008). JAGnegatively regulates KNOTTED1 LIKE HOMEOBOX expression indeveloping tomato leaves and positively regulates expression ofthe auxin-responsive genes PIN-FORMED1, AUXIN/INDOLE-3-ACETIC ACID 9 (IAA9), and IAA4, which are important for leaflet

initiation and for lamina outgrowth in tomato (David-Schwartzet al., 2009). As our results support the function of JAG asa repressor that uses the EAR motif, it will be interesting tofurther elucidate what genes/signals upstream or downstream ofJAG in the genetic regulatory networks interact directly with JAGat the molecular level.Our transformation and expression studies indicate that both

Gm-JAG1 and Gm-JAG2, which are located on homoeologouschromosomes, are functional, as indicated by the partial resto-ration of the wild-type phenotypes in the Arabidopsis jag-3mutant. Dinneny et al. (2006) showed that the functional differ-ences between JAG and its paralog NUB in Arabidopsis arecaused by slightly different tissue expression patterns. Our ex-pression analysis indicated that Gm-JAG1 and Gm-JAG2 areexpressed in a different range of tissues, suggesting sub- orneofunctionalization of Gm-JAG2. Therefore, the results fromthe previous Arabidopsis JAG paralogs are somewhat reminis-cent of how a defective mutation in one of two highly similarsoybean genes, Gm-JAG1 and Gm-JAG2, causes phenotypicdifference between mutated and wild-type soybeans.In this study, we demonstrated that the recently completed

whole-genome sequence of soybean could serve as bridginginformation to translate the information gained from a modelspecies into gene discovery and functional characterization ina crop species, soybean. We then further analyzed siliquephenotypes in order to gain insight into yield, which has beengenerally regarded as a complex trait. Direct evidence of plei-otropy for the leaflet shape and NSPP by the cloning of the lngene has fundamental implications for yield improvement, atleast for sprout specialty soybeans, by individually manipulatingthe component traits using molecular-assisted selection andprovides insight into locating the position of JAG as a repressorin genetic regulatory networks.

METHODS

Plant Materials

A BC3F2 population was developed from four self-fertilized BC3F1 hybridsmade between an elite narrow leaflet soybean (Glycine max) cultivar forsprouts (the female parent, Sowon) and a broad leaflet soybean cultivar(the male parent, V94-5152) (hereafter, referred to as the SV population). Atotal of 309 F2 individuals were previously used to establish linkage re-lationships among molecular markers, the NSPP, and the narrow leafletshape trait presumed to be determined by the ln gene (Jeong et al., 2011).

To further dissect the 66-kb genomic region detected in the previousstudy (Jeong et al., 2011), 4219 F3 seedlings derived from 162 F2 plantsheterozygous for both Ln_at004 and Ln_atre04 were grown and screenedfor recombinants between Ln_at004 and Ln_atre04. Plants with thehomozygous genotype at one marker and heterozygous genotype at theother locus were selected, and a total of 17 individuals were grown forphenotypic evaluation.

The genetic correlation between markers and leaf shape was assessedin a collection of 71 presumably diverse soybean cultivars or wild acces-sions listed in Supplemental Table 3 online. Among them, 14 variants werefurther analyzed by sequencing: Sowon, Pungsannamul, Myeongjunamul,IT182932, IT178535, IT191201, IT184014, and PI549049 collected ordeveloped in Korea, V94-5152 and Williams 82 developed in the US, andfour wild soybean accessions PI378691 (collected in the Japan), PI407290(China), PI423991 (Russia), and PI518282 (Taiwan).

Control of Fruit Patterning by Ln 4815

DNA Isolation and Marker Analysis

Young trifoliolate leaf tissues from soybean accessions were collected.Soybean genomic DNA was isolated as described previously by SaghaiMaroof et al. (1984). For quick preparation from the F2:3 plants of the SVpopulation, soybean genomic DNA was isolated using a FastDNA kit inaccordance with the manufacturer’s protocols (MP Biomedicals) froma single young leaflet. The extracted DNA was dissolved in 200 mL water,and then 2 mL of the solution was used in a 20-mL PCR to amplify DNAfragments for marker genotyping. Microsatellite and single nucleotidepolymorphism markers were analyzed as previously described (Creganet al., 1999; Jeong and Saghai-Maroof, 2004).

PCR of Genomic DNA and Sequencing

Genomic DNA isolation, PCR primer design, PCR amplification, PCRfragment purification, and sequencing of PCR fragments were conductedas described previously (Jeong and Saghai-Maroof, 2004). Primers used forPCR amplification of Gm-JAG1 and Gm-JAG2 are listed in SupplementalTable 2 online. In brief, the PCR mixture contained 20 ng of total genomicDNA, 13 PCR buffer (10 mM Tris-HCl and 50 mM KCl, pH 8.3), 2.5 mMMgCl2, 100 nM of each forward/reverse primer, 0.16 mM of each deoxy-nucleotide triphosphate, and 0.25 units of Taq polymerase in a total volumeof 20 mL. Standard PCR was conducted as follows: a denaturation step at94°C for 5 min, 34 cycles at 94°C for 30 s, 43 to 58°C for 30 s, 72°C for 30 s,and an extension step at 72°C for 5 min followed by a 4°C soak. Alignmentof the nucleotide and amino acid sequences was performed using ClustalWimplemented in BioEdit (Hall, 1999).

Vector Construct for Transformation

For the PAtJAG:gGmJAG1 vector, the JAG promoter was amplified firstfrom the Arabidopsis thaliana Ler ecotype using primers pAtJAG andXhoI-pAtJAG listed in Supplemental Table 2 online. The promoter wasthen cloned into the pENTR/D-TOPO entry vector (Invitrogen) followingthe manufacturer’s protocol. The genomic fragments of Gm-JAG1 (Ln)were amplified from soybean cultivar V94-5152 using primers XhoI-soyJAG-1 and soyJAG. The DNA fragment was then cloned into thepENTR/D-TOPO entry vector. The genomic Gm-JAG1 in V94-5152 wassubcloned into the PAtJAG entry vector using AscI and XhoI sites. ThePAtJAG:gGmJAG1 cassette was transferred to the destination vectorpHGWL7 using the attL 3 attR reaction as described in the Gatewaycloning technology instruction manual (Invitrogen). For the PAtJAG:gGmjag1 vector, the same procedure was followed using genomic DNAfrom Sowon (ln) for the genomic fragment of ln.

For the 35S:gGmJAG1 vector, the genomic DNA fragment of Ln wasamplified from V94-5152 (Ln) by PCR using primers p35S-So and SoyJAG.The resulting genomic DNA was cloned into the pENTR/D-TOPO entryvector following the manufacturer’s protocol. The attL 3 attR reactionbetween the entry vector and the destination vector pH2GW7 was per-formed to place the gGmJAG1 downstream of a cauliflower mosaic virus35S promoter as described in the Gateway cloning technology instructionmanual. For the 35S:gGmjag1 vector, the same procedure was followedusing genomic DNA from Sowon (ln) for the genomic fragment of ln.

For the PGmJAG1:gGmJAG1 vector, the JAG promoter region wasamplified first from V94-5152 by PCR using primers SoyJAG-p andSoyJAG-B. The promoter region was then cloned into the pENTR/D-TOPO entry vector. The coding region of Gm-JAG1 was amplified fromV94-5152 using a SoyJAG primer set. The resulting genomic DNA wascloned into the pENTR/D-TOPO entry vector following the manufacturer’sprotocol. The gGmJAG1 was then subcloned into the PGmJAG1 entryvector using NotI and BglII sites. PGmJAG1:gGmJAG1was transferred tothe destination vector pHGWL7 using the attL 3 attR reaction. For thePGmjag1:gGmjag1 vector, the same procedure was followed using genomic

DNA from Sowon (ln) for the genomic fragment of ln except subcloning ofthe gGmjag1 into the PGmjag1 entry vector using NotI and XbaI sites.

For the PAtJAG:gGmJAG1DEAR vector, the genomic fragment of theEAR motif–deleted Gm-JAG1 was amplified from the V94-5152 usingprimers XhoI-deltaEAR and soyJAG. This DNA fragment was then clonedinto the pENTR/D-TOPO entry vector. The deletion EAR-modified JAGwas subcloned into the pAtJAG entry vector using AscI and XhoI sites.The PAtJAG:gGmJAG1DEAR cassette was transferred to the destinationvector pHGWL7 using the attL 3 attR reaction.

For thePAtJAG:gGmJAG2 vector, the genomic fragment of Gm-JAG2,the Gm-JAG1 homoeolog, was amplified from V94-5152 using primersXho1-hm-soyJAG and hm-soyJAG. The genomic fragmentwas then clonedinto the pENTR/D-TOPO entry vector. Then, gGmJAG2 was subcloned intothe pAtJAG entry vector using AscI and XhoI sites. The Gateway cassetteof PAtJAG:gGmJAG2 was transferred into pHGWL7 by the attL 3 attRreaction.

Arabidopsis Transformation

The gene constructs were introduced into Agrobacterium tumefaciensstrain GV3101. Transgenic plants were generated by vacuum infiltrationof Arabidopsis Ler mutant jag-3 recipient plants kindly provided by E.Meyerowitz using the Agrobacterium strain GV3101 (Bechtold andPelletier, 1998), and plants were selected using resistance against theantibiotic hygromycin.

DNA Gel Blot Analysis

Eight micrograms of the genomic DNA from both V94-5152 and Sowonwere digested overnight with restriction enzymes, fractionated on 1%agarose gel, alkaline transferred onto Hybond N+ nylon membranes(Amersham Pharmacia), and probed using biotin-labeled Gm-JAG1 geneprobes amplified using SoyJAG-2and SoyJAG-2 listed in SupplementalTable 2 online. DNA probe preparation was conducted using the NickTranslation System (Invitrogen) in accordance with the manufacturer’sinstructions. The biotin-labeled DNA was then detected via chemilumines-cence using streptavidin alkaline phosphatase and CDP-Star (Applied Bio-systems). Hybridization,membranewashing, anddetectionwereall conductedin accordance with the manufacturer’s instructions. The size of the DNA bandwas compared with a 1-kb DNA ladder (Bioneer).

RNA Isolation and Expression Analysis

For analysis of the expression of Gm-JAG1 and Gm-JAG2 in differentsoybean plant tissues, total RNA was isolated from expanded leaves,young leaves, meristems, flowers, and young pods collected from threeindividuals. For the examination of Gm-JAG1 and its variants and Gm-JAG2 in the transgenic Arabidopsis jag-3 lines, total RNA was isolatedfrom Arabidopsis inflorescences. The tissues were frozen immediately inliquid nitrogen and then ground with a mortar and pestle. RNA was ex-tracted using the RNeasy plant mini kit (Qiagen). Before cDNA synthesis,all RNA samples were treated using RNase-free RQDNase (Promega). ForRT-PCR, cDNA was synthesized by one-step RT-PCR (Qiagen) usinggene-specific primers as described by the manufacturer. The PCRproducts were generated using the following primer sets: cJAG-sp forGm-JAG1 and cJAG-hsp for Gm-JAG2. The soybean Actin 11 gene wasused as an internal control for RT-PCR analysis in soybean (Jian et al.,2008), and theArabidopsis UBQ10 gene was used as an internal control inArabidopsis (Czechowski et al., 2005). The PCR products were separatedby agarose gel electrophoresis on a 1.5% gel with ethidium bromidestaining. For qRT-PCR, first-strand cDNAwas synthesized from 1 mg totalRNA in a 20-mL reaction mixture using the AccuPower RocketScript RTPreMix kit (Bioneer). All qRT-PCR reactions were performed in an ABI

4816 The Plant Cell

7900 HT sequence detection system (Applied Biosystems) using the PowerSYBRGreenPCRMasterMix kit (AppliedBiosystems) and the following two-step PCR profile: 10 min at 95°C, followed by 45 cycles of 15 s at 95°C, 30 sat 62°C, and 30 s at 72°C. PCR specificity was subjected to the machine’sstandard dissociation curve analysis. Specific gene expression was nor-malized to the internal control gene Actin 11, and a negative control withoutcDNA was also performed for each primer set. The gene expression value ofthe meristem of Ln plants was used as the control and set to 1.0. Experi-ments were performed in two biological replicates, with three technicalreplicates. The PCR primers used are listed in Supplemental Table 2 online.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: Gm-JAG1 (JX119212 toJX119213 and JX119216 to JX119226), Gm-JAG2 (JX119214 to JX119215),At-JAG (AY465924), NUB (NM_101210, LYRATE (EU490614), and Zm-JAG(NM_001153894).

Supplemental Data

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

Supplemental Figure 1. Alignment of Amino Acid Sequences ofSoybean JAG1 and Its Homoeolog JAG2, At-JAG, and NUBBIN fromArabidopsis thaliana, LYRATE from Tomato, and Zm-JAG from Maize.

Supplemental Figure 2. Silique Phenotypes Observed in ArabidopsisWild-Type Landsberg erecta, jag-3 Mutant, Three jag-3 Mutant LinesTransformed with Transgene PAtJAG:gGmJAG1, and Three jag-3Mutant Lines Transformed with Transgene PAtJAG:gGmjag1.

Supplemental Figure 3. Comparison of Phenotypes of 35S:GmJAG1,35S:Gmjag1, PGmJAG1:gGmJAG1, PGmjag1:gGmjag1, PAtJAG:gGmJAG1DEAR, and PAtJAG:gGmJAG2 Gene Constructs in theArabidopsis jag-3 Mutant.

Supplemental Figure 4. Copy Number Analysis of Gm-JAG1 andGm-JAG2.

Supplemental Figure 5. Variation in Leaflet Shape among Cultivatedand Wild Soybean Accessions.

Supplemental Figure 6. Polymorphic Sites Identified from Compar-ison of Gm-JAG1–Containing Sequences from 14 Soybean Lines.

Supplemental Table 1. Attributes of Single Nucleotide PolymorphismMarkers for Fine Mapping.

Supplemental Table 2. Primers for Sequencing, RT-PCR, and ProbeGeneration Used in This Study.

Supplemental Table 3. Leaflet Shapes of Cultivated and WildSoybean Accessions and Their Genotype at Marker Ln-AH Locus.

ACKNOWLEDGMENTS

This study was supported principally by a grant from the Next-Generation BioGreen 21 Program (PJ008124), by the Rural DevelopmentAdministration, and partly by the Korea Research Institute of Bioscienceand Biotechnology Research Initiative Program. We declare no conflictof interest. We thank Elliot Meyerowitz at the California Institute of Technologyfor providing seeds of Arabidopsis ecotype Ler and mutant jag-3. We alsothank Jieun Yun for technical assistance with qRT-PCR.

AUTHOR CONTRIBUTIONS

N.J., S.J.S., and M.K. performed the experiments. S.L., J.-K.M., and H.S.K. contributed the experimental soybean accessions and population and

designed the research. S.-C.J. designed the research and wrote thearticle.

Received September 8, 2012; revised November 27, 2012; acceptedDecember 4, 2012; published December 14, 2012.

REFERENCES

Bechtold, N., and Pelletier, G. (1998). In planta Agrobacterium-mediatedtransformation of adult Arabidopsis thaliana plants by vacuum infiltration.Methods Mol. Biol. 82: 259–266.

Bernard, R.L., and Weiss, M.G. (1973). Qualitative genetics. InSoybeans: Improvement, Production, and Uses. B.E. Caldwell, ed(Madison, WI: American Society of Agronomy), pp. 117–154.

Bowman, J.L., Alvarez, J., Weigel, D., Meyerowitz, E.M., and Smyth, D.R. (1993). Control of flower development in Arabidopsis thaliana byAPETALA1 and interacting genes. Development 119: 721–743.

Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M.,Hudson, A., and Martienssen, R.A. (2000). Asymmetric leaves1mediates leaf patterning and stem cell function in Arabidopsis.Nature 408: 967–971.

Carlson, J.B., and Lersten, N.R. (2004). Reproductive morphology. InSoybeans: Improvement, Production, and Uses, 3rd ed, H.R. Boermaand J.E. Specht, eds (Madison, WI: ASA, CSSA, and SSSA), pp. 59–95.

Coen, E.S., and Meyerowitz, E.M. (1991). The war of the whorls: Geneticinteractions controlling flower development. Nature 353: 31–37.

Cregan, P.B., Jarvik, T., Bush, A.L., Shoemaker, R.C., Lark, K.G.,Kahler, A.L., Kaya, N., Vantoai, T.T., Lohnes, D.G., Chung, J., andSpecht, J.E. (1999). An integrated genetic linkage map of thesoybean genome. Crop Sci. 39: 1464–1490.

Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible,W.R. (2005). Genome-wide identification and testing of superiorreference genes for transcript normalization in Arabidopsis. PlantPhysiol. 139: 5–17.

David-Schwartz, R., Koenig, D., and Sinha, N.R. (2009). LYRATE isa key regulator of leaflet initiation and lamina outgrowth in tomato.Plant Cell 21: 3093–3104.

Dinkins, R.D., Keim, K.R., Farno, L., and Edwards, L.H. (2002).Expression of the narrow leaflet gene for yield and agronomic traitsin soybean. J. Hered. 93: 346–351.

Dinneny, J.R., Weigel, D., and Yanofsky, M.F. (2005). A geneticframework for fruit patterning in Arabidopsis thaliana. Development132: 4687–4696.

Dinneny, J.R., Weigel, D., and Yanofsky, M.F. (2006). NUBBIN andJAGGED define stamen and carpel shape in Arabidopsis. Development133: 1645–1655.

Dinneny, J.R., Yadegari, R., Fischer, R.L., Yanofsky, M.F., andWeigel, D. (2004). The role of JAGGED in shaping lateral organs.Development 131: 1101–1110.

Domingo, W.E. (1945). Inheritance of number of seeds per pod andleaflet shape in the soybean. J. Agric. Res. 70: 251–268.

Hall, T.A. (1999). BioEdit: A user-friendly biological sequence align-ment editor and analysis program for Windows 95/98/NT. NucleicAcids Symp. Ser. 41: 95–98.

Hepworth, S.R., Zhang, Y., McKim, S., Li, X., and Haughn, G.W.(2005). BLADE-ON-PETIOLE-dependent signaling controls leaf andfloral patterning in Arabidopsis. Plant Cell 17: 1434–1448.

Hiratsu, K., Matsui, K., Koyama, T., and Ohme-Takagi, M. (2003).Dominant repression of target genes by chimeric repressors thatinclude the EAR motif, a repression domain, in Arabidopsis. Plant J.34: 733–739.

Control of Fruit Patterning by Ln 4817

Hofer, J., Turner, L., Hellens, R., Ambrose, M., Matthews, P.,Michael, A., and Ellis, N. (1997). UNIFOLIATA regulates leaf andflower morphogenesis in pea. Curr. Biol. 7: 581–587.

Jeong, N., Moon, J.K., Kim, H.S., Kim, C.G., and Jeong, S.C. (2011).Fine genetic mapping of the genomic region controlling leafletshape and number of seeds per pod in the soybean. Theor. Appl.Genet. 122: 865–874.

Jeong, S.C., and Saghai-Maroof, M.A. (2004). Detection and geno-typing of SNPs tightly linked to two disease resistance loci, Rsv1and Rsv3, of soybean. Plant Breeding 123: 305–310.

Jian, B., Liu, B., Bi, Y., Hou, W., Wu, C., and Han, T. (2008). Vali-dation of internal control for gene expression study in soybean byquantitative real-time PCR. BMC Mol. Biol. 9: 59.

Kagale, S., Links, M.G., and Rozwadowski, K. (2010). Genome-wideanalysis of ethylene-responsive element binding factor-associatedamphiphilic repression motif-containing transcriptional regulators inArabidopsis. Plant Physiol. 152: 1109–1134.

Kim, M., McCormick, S., Timmermans, M., and Sinha, N. (2003).The expression domain of PHANTASTICA determines leafletplacement in compound leaves. Nature 424: 438–443.

Lee, S.H., Park, K.Y., Lee, H.S., Park, E.H., and Boerma, H.R.(2001). Genetic mapping of QTLs conditioning soybean sprout yieldand quality. Theor. Appl. Genet. 103: 702–709.

Mandl, F.A., and Buss, G.R. (1981). Comparison of narrow and broadleaflet isolines of soybean. Crop Sci. 21: 25–27.

McHale, N.A., and Koning, R.E. (2004). PHANTASTICA regulatesdevelopment of the adaxial mesophyll in Nicotiana leaves. Plant Cell16: 1251–1262.

Norberg, M., Holmlund, M., and Nilsson, O. (2005). The BLADE ONPETIOLE genes act redundantly to control the growth and de-velopment of lateral organs. Development 132: 2203–2213.

Ohno, C.K., Reddy, G.V., Heisler, M.G., and Meyerowitz, E.M. (2004).The Arabidopsis JAGGED gene encodes a zinc finger protein that pro-motes leaf tissue development. Development 131: 1111–1122.

Ohta, M., Matsui, K., Hiratsu, K., Shinshi, H., and Ohme-Takagi, M.(2001). Repression domains of class II ERF transcriptional repressorsshare an essential motif for active repression. Plant Cell 13: 1959–1968.

Pedersen, P., and Lauer, J.G. (2004). Response of soybean yield compo-nents to management system and planting date. Agron. J. 96: 1372–1381.

Roeder, A.H., Ferrándiz, C., and Yanofsky, M.F. (2003). The role ofthe REPLUMLESS homeodomain protein in patterning the Arabi-dopsis fruit. Curr. Biol. 13: 1630–1635.

Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., and Allard,R.W. (1984). Ribosomal DNA spacer-length polymorphisms in bar-ley: Mendelian inheritance, chromosomal location, and populationdynamics. Proc. Natl. Acad. Sci. USA 81: 8014–8018.

Schmutz, J., et al. (2010). Genome sequence of the palaeopolyploidsoybean. Nature 463: 178–183.

Stanke, M., Schöffmann, O., Morgenstern, B., and Waack, S.(2006). Gene prediction in eukaryotes with a generalized hiddenMarkov model that uses hints from external sources. BMC Bio-informatics 7: 62.

Takahashi, N. (1934). Linkage relation between the genes for the formof leaves and the number of seeds per pod of soybeans. Jpn. J.Genet. 9: 208–225.

Tattersall, A.D., Turner, L., Knox, M.R., Ambrose, M.J., Ellis, T.H.N., and Hofer, J.M.I. (2005). The mutant crispa reveals multipleroles for PHANTASTICA in pea compound leaf development. PlantCell 17: 1046–1060.

Timmermans, M.C.P., Hudson, A., Becraft, P.W., and Nelson, T.(1999). ROUGH SHEATH2: A Myb protein that represses knoxhomeobox genes in maize lateral organ primordia. Science 284:151–153.

Tischner, T., Allphin, L., Chase, K., Orf, J.H., and Lark, K.G. (2003).Genetics of seed abortion and reproductive traits in soybean. CropSci. 43: 464–473.

Tsiantis, M., Brown, M.I.N., Skibinski, G., and Langdale, J.A.(1999). Disruption of auxin transport is associated with aberrant leafdevelopment in maize. Plant Physiol. 121: 1163–1168.

Tsukaya, H. (2006). Mechanism of leaf-shape determination. Annu.Rev. Plant Biol. 57: 477–496.

Weiss, M.G. (1970). Genetic linkage in soybeans. Linkage group IV.Crop Sci. 10: 368–370.

Xu, B., Li, Z., Zhu, Y., Wang, H., Ma, H., Dong, A., and Huang, H.(2008). Arabidopsis genes AS1, AS2, and JAG negatively regulateboundary-specifying genes to promote sepal and petal development.Plant Physiol. 146: 566–575.

You, M.G., Liu, Y.B., Zhao, T.J., and Gai, J.Y. (1995). Effects of leafshape on seed yield and its components in soybeans. Soyb. Genet.Newsl. 22: 66–70.

Yu, Y.H., Yu, H., Jeong, J., Park, H., Song, D., Kim, C., Kim, S., Lee,Y., Park, J., and Park, K. (2008). A General Survey of KoreanLegume Cultivars (in Korean). (Suwon, Korea: National Institute ofCrop Science).

4818 The Plant Cell

DOI 10.1105/tpc.112.104968; originally published online December 14, 2012; 2012;24;4807-4818Plant CellSoon-Chun Jeong

Namhee Jeong, Su Jeoung Suh, Min-Hee Kim, Seukki Lee, Jung-Kyung Moon, Hong Sig Kim and Is a Key Regulator of Leaflet Shape and Number of Seeds per Pod in SoybeanLn

 This information is current as of June 7, 2020

 

Supplemental Data /content/suppl/2012/12/14/tpc.112.104968.DC1.html

References /content/24/12/4807.full.html#ref-list-1

This article cites 40 articles, 17 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists