Control of petal shape and floral zygomorphyin Lotus japonicusXianzhong Feng*†, Zhong Zhao†‡, Zhaoxia Tian*†, Shilei Xu*, Yonghai Luo*, Zhigang Cai*, Yumei Wang*, Jun Yang*,Zheng Wang*, Lin Weng*, Jianghua Chen*, Leiying Zheng*, Xizhi Guo*, Jianghong Luo*, Shusei Sato§, Satoshi Tabata§,Wei Ma¶, Xiangling Cao*, Xiaohe Hu*, Chongrong Sun‡�, and Da Luo*¶�

*National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, GraduateSchool of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200032, China; ‡School of Life Sciences, Fudan University,Shanghai 200433, China; §Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan; and ¶School of Life Scienceand Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China

Communicated by Enrico Coen, John Innes Centre, Norwich, United Kingdom, January 26, 2006 (received for review November 8, 2005)

Zygomorphic flowers, with bilateral (dorsoventral) symmetry, areconsidered to have evolved several times independently in flow-ering plants. In Antirrhinum majus, floral dorsoventral symmetrydepends on the activity of two TCP-box genes, CYCLOIDEA (CYC)and DICHOTOMA (DICH). To examine whether the same molecularmechanism of floral asymmetry operates in the distantly relatedRosid clade of eudicots, in which asymmetric flowers are thoughtto have evolved independently, we investigated the function of aCYC homologue LjCYC2 in a papilionoid legume, Lotus japonicus.We showed a role for LjCYC2 in establishing dorsal identity byaltering its expression in transgenic plants and analyzing its mu-tant allele squared standard 1 (squ1). Furthermore, we identified alateralizing factor, Keeled wings in Lotus 1 (Kew1), which plays akey role in the control of lateral petal identity, and found LjCYC2interacted with Kew1, resulting in a double mutant that bore allpetals with ventralized identity to some extents. Thus, we dem-onstrate that CYC homologues have been independently re-cruited as determinants of petal identities along the dorsoventralaxis in two distant lineages of flowering plants, suggesting acommon molecular origin for the mechanisms controlling floralzygomorphy.

dorsoventral axis � floral development � keeled wings in Lotus �LjCYC2 � squared standard

F loral zygomorphy (dorsoventral asymmetry) is an evolution-ary adaptation that facilitates outcrossing by attracting pol-

linators (1–7). The phenomenal diversity in Leguminosae (Rosidclade of eudicots), the third largest family of flowering plantswith �20,000 species (8), is often explained by successful co-evolution with pollinators. In the subfamily Papilionoideae(12,000 spp.) to which Lotus japonicus (9, 10) belongs, mostspecies have specialized zygomorphic ‘‘pea’’ f lowers with threetypes of petals, which are arranged along a dorsoventral axis: asingle bilaterally symmetrical petal (standard) in the dorsalposition, two asymmetric lateral petals (‘‘wings’’) and two asym-metric ventral petals which form a ‘‘keel’’ (Fig. 1 A and B). Incontrast, Antirrhinum majus, a well studied member in Lamiales(Asterid clade) (11, 12), possesses two asymmetrical dorsalpetals, two asymmetrical lateral petals, and a single bilaterallysymmetrical ventral petal (Fig. 1 C and D). It is believed that thezygomorphy of Leguminosae has evolved separately from theLamiales (6, 13). Although the different internal symmetries ofthe counterpart petals in L. japonicus and A. majus are consistentwith the independent evolution of zygomorphy in these twolineages, both are a response to a dorsoventral axis, suggestingthat there could be a divergence in the make up of the mecha-nism to determine the axis in the control of zygomorphicdevelopments in the two distantly related species.

In A. majus, the development of zygomorphic flower requires theactivity of two TCP-box genes (14), CYC and DICH (11, 12), whosefunction is mediated through an interaction with some specific MYB

genes (15–17). When both CYC and DICH are mutated, morepetals and stamens are developed in the dorsal region, and all petalsresemble the shape of ventral petal. Thus, CYC and DICH couldhave a dual role in the control of zygomorphic development: anearly one affecting primordium initiation and controlling floralasymmetry, and a later one affecting organ asymmetry and othermorphological characters (11, 12). It has been further proposed thatthe petal asymmetry arises through a series of steps in which theexpression patterns of CYC and DICH are progressively elaboratedand maintained (12), and thus development of floral and petalasymmetries are closely related. Because TCP-box genes have beenfound widely in higher plant genomes (7, 14), it is possible to testwhether CYC like genes are involved in the development ofzygomorphy in different species. In some close relatives of Antir-rhinum, several CYC orthologues have been found to play a similarrole in the control of floral symmetry (18, 19), but other studiessuggest that the bilaterally symmetrical flowers in some families ofthe same Asterid clade might not require orthologues or functionalanalogues of CYC or DICH (6, 20). So far, because of lack of anamenable experimental system, most studies have relied on iden-tification of CYC homologues and investigation of their expressionpatterns, and little robust functional analysis in the distantly relatedspecies has been reported.

In legumes, a number of CYC-like genes have been isolatedand found to have undergone repeated duplication events,suggesting that they might have divergent functions (21, 22). Toinvestigate the molecular mechanisms underlying the develop-ment of different zygomorphic flowers among angiosperms, weexplored a model legume, Lotus japonicus (9, 10), and examinedwhether the CYC homologues also play a role in the control off loral asymmetry in this papilionoid legume. We demonstratedthat a CYC-like gene in L. japonicus, LjCYC2, has a dorsalizingactivity during petal development, similar to CYC and DICH,even though the zygomorphy of Leguminosae is believed to haveevolved separately from the Lamiales (1, 6, 7, 13). Furthermore,we identified a lateralizing factor, keeled wings in Lotus 1 (Kew1),which plays a key role in the control of lateral petal identity, andfound that LjCYC2 interacted with Kew1, resulting in a doublemutant bearing all petals with ventralized identity to a differentextent. Our data show that CYC homologues have been inde-pendently recruited to the control of f loral zygomorphy indistantly related lineages, suggesting that their dorsal activityshould be a primary and synapomorphic function during theevolution of dorsoventral specification.

Conflict of interest statement: No conflicts declared.

See Commentary on page 4801.

†X.F., Z.Z., and Z.T. contributed equally to this work.

�To whom correspondence may be addressed. E-mail: dluo@sibs.ac.cn or kmcao@fudan.edu.cn.

© 2006 by The National Academy of Sciences of the USA

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ResultsInflorescence and Floral Development in L. japonicus. L. japonicus(Gifu B-129) (9) is a perennial herb. When starting reproductivegrowth, each shoot becomes a primary inflorescence (I1), andproduces a secondary inflorescence meristem (I2) at each node.Floral meristems are generated from I2. We divided I2 devel-opment into seven stages, from I2-1 to I2-7 (Fig. 1 E–K). One I2meristem initiates from the axil of the compound leaf at eachnode (I2-1 and I2-2, Fig. 1 E and F). Bract primordia initiate atI2-3 (Fig. 1G) and become visible at I2-4 when the meristem ofI2 ceases activity and becomes compressed (Fig. 1H). Then,normally three floral meristems are partitioned sequentiallyfrom I2 (Fig. 1 I and J) and finally I2 differentiates, becomingcovered by trichomes (Fig. 1K). The development of floraldorsoventral asymmetry can be observed at the very beginningof floral development when the floral organs initiate in aunidirectional order (23), and the dorsoventral axis becomesevident (Fig. 1K): organ primordia in the dorsal region develop

more slowly than those in the ventral region. During late floraldevelopment, three types of petals develop along the dorsoven-tral axis, and a key morphological landmark is the differentiationof the vascular veins in petals. The various shapes of differentpetals are distinctive (Fig. 1L) when the primary veins initiate inall petals. Then, the characteristic shapes of dorsal, lateral, andventral petals become visible (Fig. 1M) when the initiation ofminor veins and freely ending veinlets begins and the differen-tiation of epidermal cell types commences. Further elaborationof petal structures continues, giving rise to mature petals withblades and claws in dorsal petals and blades and stipes in lateraland ventral petals (Fig. 1N), whereas various representative celltypes are generated in the epidermal layers of different petals(see Fig. 4 H–J).

CYC Homologues in L. japonicus. Four TCP-box genes, LjCYC1,LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos.

Fig. 1. Development and comparison of zygomorphic flowers. Zygomorphicflowers and petals of L. japonicus (A and B) and Antirrhinum (C and D) areshown. In the case of Antirrhinum, only the lobes of petals are shown. Lineswith arrow show the direction of floral dorsoventral axis; triangle indicatesthe keel; D, V, direction of the floral dorsoventral axis. Red broken linesindicate internal symmetry of different petals; DP, dorsal petal; LP, lateralpetal; VP, ventral petal. (Scale bars, 1.0 cm.) (E–K) Developmental stages of I2in L. japonicus Gifu B-129. At stage 1 (I2-1), I2 initiates in the axil of compoundleaf in I1 (E). When all floral meristems are developed at I2-7, the unidirectionaldevelopment of floral organs manifests the direction of the dorsoventral axis(K, lines with arrow). Star, degenerate meristem of I2; triangle, bract primor-dium; F, floral meristem. (Scale bars, 50 �m.) (L–N) The shapes of dorsal, lateral,and ventral petals in the wild type at late developmental stages. The maturedorsal petal processes blade (triangle) and claw (arrow), and the lateral andventral petals have blades (triangles) and stipes (arrows). (Scale bars, 1.0 mm.)

Fig. 2. Molecular and phylogenetic analysis of LjCYC2 and identification of amutant allele. (A) Alignment of putative protein sequences of LjCYC2 and CYC.TCP domain is underlined with straight lines and R domain is underlined withwaved lines (7). Triangle indicates position of the intron in LjCYC2. (B) A pointmutation (blue) occurred at the intron (red) splicing site of LjCYC2 in squ1, whichconsequently gives rise to a putative shorter protein with five new amino acids attheCterminus. (C)Theunsplicingof intronin squ1wasconfirmedbyRT-PCRusingtwoprimersnestedtotheintron.Thetranscriptfrom squ1 isunspliced, larger (380bp) than the one from wild type (150 bp), and LjUbi is used as the control fortemplate quantity. (D) Unrooted phylogram of protein NJ analysis of TCP domainregion suing MGA3.1 (24). Branches with bootstrap support (1,000 replicates) �

50% are indicated for each branch. All of the TCPs genes are from Arabidopsis(25); CYC and DICH, Antirrhinum; LCYC, Linaria vulgaris; LjCYCs, L. japonicus;PCFs, rice; TB1, maize.

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DQ202475, DQ202476, DQ202477, and DQ202478), wereisolated from L. japonicus. Phylogenetic analysis placed threeof them, LjCYC1, LjCYC2, and LjCYC3, in a close cladeincluding CYC and DICH (Fig. 2D), which correspond to theLEGCYC I (LjCYC1 and LjCYC2), and LEGCYC II (LjCYC3)genes, respectively (21, 22). RNA in situ hybridizations wereconducted to analyze their expression patterns. It was foundthat LjCYC1 was expressed in I1 and dorsal regions of f loralprimordia. Expression of LjCYC3 could not be detected byRNA in situ hybridization, and transcripts of LjCYC5 were

located in young leaves and at the base of I2 (data not shown).Therefore, we focus on LjCYC2, which was the most similar toCYC in expression pattern (Figs. 2 A and 3 C–F). However, incontrast to the expression pattern of CYC, which was onlyobserved in the dorsal region of f loral meristem and dorsalf loral organs, the earliest expression of LjCYC2 was found atthe margin between I1 and I2 (Fig. 3A), and also becamedetectable in the center of the I2 meristem at I2-4, when itbegan to differentiate (Figs. 1H and 3B). Later, LjCYC2 wasexpressed in the dorsal region of f loral meristems (Fig. 3 C andD), and then persisted in the dorsal organs of developingf lowers, including the dorsal sepal, petal and stamen primordia(Fig. 3 E and F). The asymmetrical expression pattern ofLjCYC2 in the f loral meristem indicates that it could play asimilar role to CYC in the control of f loral dorsoventraldevelopment in L. japonicus, whereas its expression in thedeveloping inf lorescence meristem suggests a multifunctionrole during f loral development.

Effects of Altered Expression of LjCYC2 in Transgenic L. japonicus. Totest its function, the effects of reduced or constitutive expressionof LjCYC2 were investigated in transgenic plants (Fig. 5 andSupporting Text, which is published as supporting information onthe PNAS web site). All transgenic plants expressing LjCYC2constitutively showed a specific effect in the lateral and ventralpetals. They displayed abnormal petal shapes and cell types inthe adaxial epidermal layer to varying extents: the lateral petalsbecame more symmetrical and more like dorsal petals, possess-ing dorsal-like conical cells mixed with jigsaw puzzle-shaped; the

Fig. 3. Expression pattern of LjCYC2. (A–F) Longitudinal and transversesections through the main apex of wild type were hybridized with LjCYC2antisense RNA probes labeled with digoxigenin-UTP. The transcript-specifichybridization signal is visualized in blue. (G and H), Ectopic expression ofLjCYC2 in the inflorescence (G) and floral meristems (H) in 35S::LjCYC2 trans-genic line SH3011. D(or V)Se, dorsal (or ventral) sepal primordium; D(or V)P,dorsal (or ventral) petal primordium; D(or V)St, dorsal (or ventral) stamen; C,carpel primordium. (Scale bars, 50 �m.)

Fig. 4. Flowers of the wild-type (L. japonicus, Gifu B129), transgenic, and mutant plants. (A–G) Front and side views of flowers and different petals are shown.(Scale bar, 0.5 cm.) (A) Wild-type flower. Arrow indicates that two ventral petals form a keel, and part of the ventral petals are fused at their edges. (B) A typicaltransgenic flower of sense LjCYC2 transgenics. Arrow indicates the more symmetrical lateral petal. (C) A typical flower of antisense LjCYC2 transgenics. Starindicates the keeled-wing lateral petal. (D) A flower of squ1. Arrows indicate the squared shape of dorsal petal. (E) A flower of kew1. Star indicates thekeeled-wing lateral petal. (F) A flower of the squ1kew1 double mutant. Arrow indicates where the dorsal petal was cut so as to make it flat and star indicatesthe keeled-wing lateral petal, whose size is larger than its counterpart in kew1. (G) A flower of transgenic line with GFP-tagged sense LjCYC2 (SH0578). Arrowsindicate the more symmetrical lateral and ventral petals FF, front view of flowers; FS, side views of flowers. (Scale bar, 0.5 cm.). (H–Y) The representative epidermalcells from different petals in the wild type and mutants. The wild type has conical cells on the dorsal petal, jigsaw puzzle-shaped cells on the lateral andwax-covered rectangle cells on the ventral petal (H–J). In SH3011, epidermal cells at all petals are larger than the ones in the wild type, and the conical shapedcells with the jigsaw puzzle-shaped appear on the lateral and ventral petals (K–M). In squ1, the epidermal cells on the dorsal petal display a mixed character ofboth conical and jigsaw puzzle-shaped (N–P). In kew1, the cells in the lateral petals resemble the ventral petals (Q–S). In the squ1kew1 double mutant, cells withventral identity can be found on all petals (T–V). In SH0578, nearly all of the epidermal cells in all petals are conical one (W–Y) (Scale bar, 10 �m in H–V and 50�m in W–Y.)

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ventral petals were also more symmetrical, displaying patches ofabnormal conical cells, whereas the dorsal petal maintained thewild-ype shape and cell type (Fig. 4 B and H–J). This findingindicates that the ectopic expression of LjCYC2 can confer adorsalizing effect. This effect was greatly enhanced in the GFPtagged LjCYC2 line (SH0578), with all of the petals fullydorsalized and the normal dorsoventral asymmetry abolished(Fig. 4 G and W–Y). On the other hand, the antisense transgenicplants often displayed a phenotype with abnormal lateral petalswhich resembled the ventral in both petal shape (Fig. 4C) andepidermal cell type (data not shown), suggesting that reducedLjCYC2 activity leads ventralization. Therefore, the dorsalizingeffect of LjCYC2 on petal identity was revealed in both sense andantisense transgenic plants. However, altered LjCYC2 activityalso gave rise to other morphological alterations, such as alteredinflorescence and floral structures (Fig. 5 B and C). In sensetransgenic lines, I1 terminated with shortened internodes wasobserved frequently (Fig. 5B), and in primary antisense trans-genic plants, two flowers were frequently fused to various extents

(Fig. 5C), although the variable phenotypes were weakened andvanished in succeeding generations. These data indicate that theprecise expression pattern of LjCYC2 is important for floraldevelopment, suggesting other roles for LjCYC2, which could bedistinct from its dorsalizing function.

Identification of an LjCYC2 Mutant Allele, squ1. A large scale mu-tagenesis experiment was conducted in L. japonicus (a total of�50,000 M2 families) to screen for mutations with abnormalfloral symmetry. Several single recessive mutants with abnormalpetal shape were obtained. One mutant was named squaredstandard 1 for the abnormal shape of the dorsal petal (Fig. 4D).squ1 seems to specifically affect the development of dorsal petalwithout other notable phenotypes, and its effect on petal de-velopment begins during initiation of the primary vein in thepetal (data not shown). Genetic analysis showed that squ1co-segregated with a molecular marker for the LjCYC2 locus inchromosome 2. Sequencing data confirmed that squ1 carried apoint mutation at the splicing site of the intron in LjCYC2 (Fig.2B), which gave rise to an abnormal transcript with lowerexpression level (Fig. 2C). squ1 has the capacity to encode aprotein, in which the seventeen C-terminal amino acids arereplaced by five others (Fig. 2C). This finding suggests that squ1is a mutant allele of LjCYC2. Apart from its phenotype on theshape of dorsal petals, squ1 also has a specific effect on theepidermal cell shape in the dorsal petal. The cells do not all havethe typical conical morphology, but include jigsaw puzzle-shapedcells characteristic of the lateral petals in the wild type (Fig. 4N–P), indicating a correlation between the abnormal shape ofsqu1 dorsal petal and the reduction of dorsal identity. Therefore,the mutant phenotype of ljcyc2 is in agreement with transgenicdata, confirming that LjCYC2 has a specific function in thecontrol of dorsal identity. However, squ1 did not display anyother detectable phenotype apart from the abnormal dorsalpetal, in contrast to the range of phenotypes in transgenic plantswith either sense or antisense transgene of LjCYC2.

Isolation of the kew1 Mutant. Another mutation only affects theappearance of the lateral petal and is named keeled wing in Lotus1 (kew1) for its similar phenotype to the mutant keeled wing (k)in pea (24, 25), whose flowers bear the abnormal lateral petalswith the shape mimicking keel petals. In kew1, the lateral petalsresemble the ventral in both petal shape and epidermal cell types(Fig. 4 E and Q–S), but the unidirectional initiation of floralorgan primordial (23), as well as the early development of lateralpetals up to the stage when the primary veins are initiated (Fig.1L), are the same as the wild type (data not shown). This findingsuggests that Kew1 is a specific factor in the control of lateralpetal identity and functions at a late stage during petal devel-opment. Our mapping experiment positioned kew1 in the shortarm of chromosome 5, where it shares a macro synteny with theregion containing k in pea (data not shown), suggesting that kew1and k could be the same genetic locus controlling lateral petalidentity in papilionoid legumes.

Interaction Between LjCYC2 and Kew1. We tested for interactionbetween LjCYC2 and Kew1 by crossing squ1 with kew1, becausemutants of both genes displayed effects on the shape and identityof dorsal and lateral petals, respectively. The squ1kew1 doublemutant, identified in the F2 population, had enhanced ventralphenotypes on dorsal and lateral petals, leading to all petalsbecoming ventralized to a different extent: the dorsal petal wasmuch smaller and did not expand outward (Fig. 4F), with someadaxial epidermal cells possessing the characters of normalventral petals (Fig. 4T); the lateral petals were ventralized butlarger than in kew1 (Fig. 4F), and the cell size and morphologywere also ventralized (Fig. 4 R and U). Thus, LjCYC2 and Kew1have roles which are not limited to only the dorsal or lateral

Fig. 5. Comparison of primary inflorescence and secondary inflorescence (I2)between wild type and transgenic plants of L. japonicus. (A) In wild type, I2develops from the axil of the compound leaf at each node (arrows) and usuallyproduces two flowers with diadelphous stamens (triangle). An internode isindicated with a star. (B) In sense transgenic line SH3011, a few abnormalnodes with shorter internodes (stars) are normally generated before one or afew I2 (arrows) replace I1. All stamens are separate from each other in thestrong phenotype flowers with additional bract leaflets are generated. (C)Two I2 (indicated with two arrows) from the same node are occasionallyobserved (star) and two flowers are frequently fused in various extents atantisense transgenic plants (line SH3001). St, stamen; Br, bract; Fu, fusionflowers. (Scale bar, 1.0 cm.) (D) Expression levels of LjCYC2 in the wild-type andtransgenic lines. The transcription of LjCYC2 cannot be detected in the leavesof wild-type and antisense transgenic lines (SH3001 and SH3005), and it isreduced in floral tips of antisense transgenic lines, but accumulated at highlevel in both leaves and floral tips of the sense transgenic line (SH3011 andSH3012). NDT, not detected; #, standard deviation.

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petals, respectively, indicating that both genes are used togetherin the dorsoventral mechanism to determine petal identities. Onthe other hand, there is no defect in floral organ initiation orinflorescence development in the squ1kew1 double mutant (datanot shown), suggesting that both LjCYC2 and Kew1 could havespecific role in the control of petal identity along dorsoventralaxis.

DiscussionIn this study, we exploited a model legume, L. japonicus, toconduct a robust functional analysis of a CYC-like gene, LjCYC2,during zygomorphic floral development. We have demonstratedthat LjCYC2, similar to CYC in A. majus, has an asymmetricexpression pattern in floral meristems and a dorsalizing activityon petal identity, even though the zygomorphy of Leguminosaeis believed to have evolved separately from the Lamiales (1, 6, 7,13). A recent study has shown that the expression pattern of anLjCYC2 orthologue is altered in the legume Cadia, which istypical of a series of unrelated papilionoid genera with unusual,more or less radially symmetrical f loral morphologies, support-ing a role for CYC homologues in the evolution of floral noveltyin legumes (H. L. Citerne, R. T. Pennington, and Q. C. B. Cronk,personal communication). Thus, these data reveal that CYChomologues have been recruited in Leguminosae and play animportant role in the development of dorsoventral asymmetry.

It may not be coincidence that both LjCYC2 and CYC conferdorsal activities in the two distantly related lineages. Although theprecise functions of CYC and LjCYC2 on the development ofzygomorphy need to be clarified further, mutants of both genesdisplay similar effects on petals at the cellular level, such asdetermining the epidermal cell types, size, and shape. This findingis in agreement with the general role of the TCP-box genes, whichis linked to the regulation of cell division and proliferation (26–28).It suggests that a conserved function shared by TCP-box genes isimportant for the dorsal activity of both LjCYC2 and CYC. In theRosid Arabidopsis, which has radially symmetric flowers, the closesthomologue to CYC and LjCYC2, TCP1, displays an asymmetricexpression pattern transiently in the dorsal domain of floral mer-istems, as well as in the adaxial region of axillary shoot meristems(29). It has been speculated that an ancestral asymmetry had beencreated in a common ancestor of A. majus and Arabidopsis by aCYC�TCP1-related gene, which presumably had the radially sym-metrical flower, and further evolution of this gene, such as changesin timing or levels of expression and interactions with the targetgenes, may underlie repeated and independent recruitment ofCYC-like genes to control of zygomorphic floral development (29,30). Consistently, our finding about the dorsalizing function ofLjCYC2 indicates that the similar evolutionary course of CYC-likegenes have occurred independently in Leguminosae. Thus, theCYC-like genes could help to define the dorsoventral axis through-out angiosperms and there could be a common molecular origin forthe mechanisms controlling floral zygomorphy. However, althoughboth CYC and LjCYC2 possess dorsalizing activity, they bring aboutthe distinct zygomorphic flowers in A. majus and L. japonicus,respectively, which produce different internal symmetry in theircounterpart dorsal and ventral petals (Fig. 1 B and D), suggestingthat LjCYC2 and CYC could have been integrated into differentgenetic networks with divergent roles controlling the zygomorphicdevelopment. It is likely that the evolution of dorsoventral axis tocontrol of zygomorphic flowers does not only depend on the extentto which the ancestral dorsalizing activity has been elaborated, butalso rely on the way how it was responded in each zygomorphicclade. Thus, the dorsalizing activity should be a primary andsynapomorphic function being recruited independently into themechanism to control the dorsoventral axis in different lineages.

There are some hints to how the dorsoventral axis might haveevolved in the distantly related species independently. Leguminosaeconsists of three subfamilies, Caesalpinioideae, Mimosoideae, and

Papilionoideae. Caesalpinioideae is a paraphyletic assemblage fromwhich the monophyletic Mimosoideae and Papilionoideae arose.Caesalpinioideae flowers are variable in morphology, althoughoften subtly zygomorphic, whereas Mimosoideae has mostly specieswith actinomorphic flowers, and most species in Papilionoideaedisplay prominent zygomorphic ‘‘pea’’ flowers. It has been shownthat the CYC-like genes have undergone two duplication events: anearly one giving rise to LEGCYC I and LEGCYC II genes couldhave occurred before the evolution of Papilionoideae, and a laterone resulting in LEGCYC IA and LEGCYC IB have occurredduring the early diversification of Papilionoideae (21, 22). So far,only LEGCYC IB genes, such as LjCYC2 in L. japonicus and itshomologue in pea (unpublished data), have been found to possessdorsalizing activity and control the development of dorsal petals. Incontrast to the expression pattern of CYC, LjCYC2 in L. japonicusis expressed transiently in the inflorescences (Fig. 3 A–F), andaltered LjCYC2 expression in transgenic plants had effects on boththe development of inflorescences and flowers (Fig. 5C), suggestingmultiple developmental roles of LjCYC2 that are not shared withCYC in A. majus. Although a mutant of LjCYC2 (squ1) affectedonly the identity of dorsal petals (Fig. 4D), it is possible that squ1is a weak mutation giving rise to a partially functional protein,because its transcript is detectable and capable of encoding amutant protein with the TCP and R domains intact. Alternatively,the function of LjCYC2 in the inflorescences may be redundant,because the LEGCYC IA gene LjCYC1 is also expressed transientlyin inflorescence meristems before it is asymmetrically expressed inthe dorsal region of floral meristem. It has been shown that petalasymmetry in A. majus could arise through a series of steps in whichthe expression patterns of CYC and its homologue DICH areprogressively elaborated and maintained from early floral meristeminitiation through late stages of development, which should poten-tially reflect a sequence of evolutionary events (12). Similarly, thedynamic expression pattern of LjCYC2 and LjCYC1 in the inflo-rescences might be necessary for elaborating the dorsoventral axisin floral meristems, and register the evolutionary course of theirancestor in Leguminosae. It is possible that the duplication ofCYC-like genes and their functional divergence are generally im-portant for the inherent dorsalizing activity of their ancestor to beelaborated and responded, so that the dorsoventral axis has beenevolved to produce the prominent zygomorphic flowers in differentlineages. Further analysis of the expression pattern and function ofdifferent CYC-like genes should provide more information abouthow gene duplication could contribute to the elaboration of dor-soventral axis and the development of zygomorphy among angio-sperms.

A genetic factor, Kew1 in L. japonicus, was identified in this study,the mutant of which affects the development of lateral petal only,resulting in the lateral petal resembling the ventral in both petalshape and epidermal cell types (Fig. 4 E and Q–S). Because only theidentity of lateral petals is altered and no other defect has beenfound in the mutant kew1, Kew1 should have a specific role in thecontrol of lateral petal identity. Although their relationship needsfurther investigation, the similar phenotypes of the mutant kew1and k suggest that Kew1 and K might be the same genetic compo-nent in the same pathway, and represent a lateralizing factor. Wefound that LjCYC2 could interact with Kew1, and the doublemutant bore ventralized petals. The enhanced phenotype ofsqu1kew1 double mutant in the dorsal petal indicates that theactivity of Kew1 could also be needed for the development of dorsalpetals, suggesting that there is a link between the dorsalizing andlateralizing activities which should be important for the elaborationof dorsoventral axis. This possibility should be properly addressedwith the cloning of Kew1 and K from L. japonicus and pea in thenear future.

Both single mutant, kew1 and squ1, and the double mutantdisplay defects on the development of dorsal and�or lateral petals,and their malfunction appears in the late stages of floral develop-

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ment when primary veins are initiated in petals (Fig. 1M). It hasbeen reported that morphologic differentiation of floral organsalong the dorsoventral axis in papilionoid legumes occurs in the latestages of floral ontogeny (31). The specific activities and interactionof LjCYC2 and Kew1 to control the identities of dorsal and lateralpetals in the late stage of floral development could provide themolecular basis for the characteristic floral ontogeny in legumes.On the other hand, the development of dorsoventral axis is evidentfrom the beginning of floral organ initiation, suggesting that therecould be a heterochronic role for the dorsalizing and lateralizingactivities in the control the development of dorsoventral axis andthe petal identity respectively. In A. majus, the cyc mutant displayspartial, and the cycdich double mutant complete, loss of dorsalizedpetals, with an increased number of dorsal organs, suggesting thatCYC�DICH have an early function to determine the floral organnumbers (11, 12). In contrast, although all petals are ventralized toa different extent, there is no increase of floral organ numbers inthe squ1kew1 double mutant, indicating that the LjCYC2 does notplay the same role as the one of CYC in the control of floral organinitiation. These data further suggest that the independent recruit-ment of CYC-like genes within each zygomorphic clade might resultin the acquisition of different functions, which could in turn haveimplication for the divergence of the mechanisms and account forthe distinct zygomorphy between different lineages.

Materials and MethodsPlant Material and Growth Conditions. L. japonicus ecotypes GifuB129 and Miyakojima (MG-20) were used in this study. Allplants were grown at 20–22°C with a 16-h light�8-h darkphotoperiod at 150 mE�m�2�s�1.

Two mutants, kew1 and squ1 were isolated from a � raymutagenized M2 population in the Gifu B129 background. Afterseveral backcrosses, in which 3:1 ratios of the wild type andmutant were observed in the segregating populations, lineSH0389 for kew1 and line SH0120 for squ1 were established.

Gene Cloning and RT-PCR. Fragments of CYC-like genes wereamplified from L. japonicus by using degenerate oligonucleotideprimers Pcyc1 and rPcyc2 (sequences of all primers are given inSupporting Text) in 5�-RACE using the 5�-RACE system(GIBCO�BRL). RACE products were used to screen a genomiclibrary (32). A pair of primers around the intron of LjCYC2

(SL1716 and SL1717) was used to detect the splicing in both wildtype and mutant.

Phylogenetic Analysis. Protein distance analysis was carried out byusing MEGA version 3.1 (33) by the full length of proteinsequences from GenBank according to refs. 11, 12, 14, 18, 29,and 34. Distance matrices used the PAM-Dayhoff model ofamino acid substitution; the phylogenetic tree was constructedwith the neighbor-joining method, and bootstrap analyses used1,000 resampling replicates. To simplify the analysis, only thefull-length TCP protein sequences of some representative andparticular interest TCP-box genes were used.

Microscopy. Tissue for in situ hybridization was fixed overnight in4% (wt�vol) paraformaldehyde in phosphate buffer, pH 7.0, andembedded in Paraplast (Sigma). Nonradioactive in situ hybrid-ization was performed essentially as described (35). Scanningelectron microscopy was performed on plastic replicas as de-scribed earlier (36). Contrast and color balance were adjusted byusing PHOTOSHOP 8.0 (Adobe, Mountain View, CA).

Transgenic Plants. Transformation of L. japonicus was carried outas described (9). Details of transgenic constructions and trans-genic lines are described in Supporting Text. The alteration ofLjCYC2 expression level in some lines were confirmed byreal-time PCR (Supporting Text).

Mapping. kew1 was mapped by using an F2 mapping population,resulting from a cross between kew1 (from the Gifu B129background) and Miyakojima (MG-20). In total, 6,000 F2 indi-viduals were analyzed with markers flanking kew1. squ1 wasmapped with a F2 mapping population whose size was 160 plants.Two primers (LJSSR0527 and LJSSR0528) were used as the SSRmarkers for the genomic region containing LjCYC2.

We thank A Hudson, C. Kidner, R. T. Pennington, Q. C. Cronk,D. Bradley, M. Ambrose, P. Cubas, and E. Coen for their help and criticalcomments on the manuscripts; two anonymous reviewers for providinghelp and advice; and Z. Xu, J. Li, Y. Xue, G. Wang, Y. Tian, H. Lin, Y.Wang, and S. Ge for their genuine encouragement and support for thisexperiment. This work was supported by National Natural ScienceFoundation of China Grant 30430330 and Ministry of Science andTechnology of the People’s Republic of China Grant 2003AA222030.

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