Control of petal shape and floral zygomorphy in japonicus

Xianzhong 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‡ʈ, and Da Luo*¶ʈ

*National Key Laboratory of Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Graduate School 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 Science and 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, are genes (15–17). When both CYC and DICH are mutated, more considered to have evolved several times independently in flow- petals and stamens are developed in the dorsal region, and all petals ering . In Antirrhinum majus, floral dorsoventral symmetry resemble the shape of ventral petal. Thus, CYC and DICH could depends on the activity of two TCP-box genes, CYCLOIDEA (CYC) have a dual role in the control of zygomorphic development: an and DICHOTOMA (DICH). To examine whether the same molecular early one affecting primordium initiation and controlling floral mechanism of floral asymmetry operates in the distantly related asymmetry, and a later one affecting organ asymmetry and other Rosid clade of , in which asymmetric flowers are thought morphological characters (11, 12). It has been further proposed that to have evolved independently, we investigated the function of a the petal asymmetry arises through a series of steps in which the CYC homologue LjCYC2 in a papilionoid legume, Lotus japonicus. expression patterns of CYC and DICH are progressively elaborated We showed a role for LjCYC2 in establishing dorsal identity by and maintained (12), and thus development of floral and petal altering its expression in transgenic plants and analyzing its mu- asymmetries are closely related. Because TCP-box genes have been tant allele squared standard 1 (squ1). Furthermore, we identified a found widely in higher plant genomes (7, 14), it is possible to test lateralizing factor, Keeled wings in Lotus 1 (Kew1), which plays a whether CYC like genes are involved in the development of key role in the control of lateral petal identity, and found LjCYC2 zygomorphy in different species. In some close relatives of Antir- interacted with Kew1, resulting in a double mutant that bore all rhinum, several CYC orthologues have been found to play a similar petals with ventralized identity to some extents. Thus, we dem- role in the control of floral symmetry (18, 19), but other studies onstrate that CYC homologues have been independently re- suggest that the bilaterally symmetrical flowers in some families of cruited as determinants of petal identities along the dorsoventral the same Asterid clade might not require orthologues or functional axis in two distant lineages of flowering plants, suggesting a analogues of CYC or DICH (6, 20). So far, because of lack of an common molecular origin for the mechanisms controlling floral amenable experimental system, most studies have relied on iden- zygomorphy. tification of CYC homologues and investigation of their expression patterns, and little robust functional analysis in the distantly related dorsoventral axis ͉ floral development ͉ keeled wings in Lotus ͉ species has been reported. LjCYC2 ͉ squared standard In legumes, a number of CYC-like genes have been isolated and found to have undergone repeated duplication events, loral zygomorphy (dorsoventral asymmetry) is an evolution- suggesting that they might have divergent functions (21, 22). To Fary adaptation that facilitates outcrossing by attracting pol- investigate the molecular mechanisms underlying the develop- linators (1–7). The phenomenal diversity in Leguminosae (Rosid ment of different zygomorphic flowers among angiosperms, we clade of eudicots), the third largest family of flowering plants explored a model legume, Lotus japonicus (9, 10), and examined with Ϸ20,000 species (8), is often explained by successful co- whether the CYC homologues also play a role in the control of evolution with pollinators. In the subfamily Papilionoideae floral asymmetry in this papilionoid legume. We demonstrated (12,000 spp.) to which Lotus japonicus (9, 10) belongs, most that a CYC-like gene in L. japonicus, LjCYC2, has a dorsalizing species have specialized zygomorphic ‘‘pea’’ flowers with three activity during petal development, similar to CYC and DICH, types of petals, which are arranged along a dorsoventral axis: a even though the zygomorphy of Leguminosae is believed to have single bilaterally symmetrical petal (standard) in the dorsal evolved separately from the Lamiales (1, 6, 7, 13). Furthermore, position, two asymmetric lateral petals (‘‘wings’’) and two asym- we identified a lateralizing factor, keeled wings in Lotus 1 (Kew1), metric ventral petals which form a ‘‘keel’’ (Fig. 1 A and B). In which plays a key role in the control of lateral petal identity, and contrast, Antirrhinum majus, a well studied member in Lamiales found that LjCYC2 interacted with Kew1, resulting in a double (Asterid clade) (11, 12), possesses two asymmetrical dorsal mutant bearing all petals with ventralized identity to a different petals, two asymmetrical lateral petals, and a single bilaterally extent. Our data show that CYC homologues have been inde- symmetrical ventral petal (Fig. 1 C and D). It is believed that the pendently recruited to the control of floral zygomorphy in zygomorphy of Leguminosae has evolved separately from the distantly related lineages, suggesting that their dorsal activity Lamiales (6, 13). Although the different internal symmetries of should be a primary and synapomorphic function during the the counterpart petals in L. japonicus and A. majus are consistent evolution of dorsoventral specification. with the independent evolution of zygomorphy in these two lineages, both are a response to a dorsoventral axis, suggesting that there could be a divergence in the make up of the mecha- Conflict of interest statement: No conflicts declared. nism to determine the axis in the control of zygomorphic See Commentary on page 4801. developments in the two distantly related species. †X.F., Z.Z., and Z.T. contributed equally to this work. In A. majus, the development of zygomorphic flower requires the ʈTo whom correspondence may be addressed. E-mail: [email protected] or kmcao@ activity of two TCP-box genes (14), CYC and DICH (11, 12), whose fudan.edu.cn. function is mediated through an interaction with some specific MYB © 2006 by The National Academy of Sciences of the USA

4970–4975 ͉ PNAS ͉ March 28, 2006 ͉ vol. 103 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600681103 Downloaded by guest on September 25, 2021 SEE COMMENTARY

Fig. 1. Development and comparison of zygomorphic flowers. Zygomorphic flowers and petals of L. japonicus (A and B) and Antirrhinum (C and D) are shown. In the case of Antirrhinum, only the lobes of petals are shown. Lines Fig. 2. Molecular and phylogenetic analysis of LjCYC2 and identification of a with arrow show the direction of floral dorsoventral axis; triangle indicates mutant allele. (A) Alignment of putative protein sequences of LjCYC2 and CYC. the keel; D, V, direction of the floral dorsoventral axis. Red broken lines TCP domain is underlined with straight lines and R domain is underlined with indicate internal symmetry of different petals; DP, dorsal petal; LP, lateral waved lines (7). Triangle indicates position of the intron in LjCYC2.(B) A point mutation (blue) occurred at the intron (red) splicing site of LjCYC2 in squ1, which petal; VP, ventral petal. (Scale bars, 1.0 cm.) (E–K) Developmental stages of I2 consequently gives rise to a putative shorter protein with five new amino acids at in L. japonicus Gifu B-129. At stage 1 (I2-1), I2 initiates in the axil of compound the C terminus. (C) The unsplicing of intron in squ1 was confirmed by RT-PCR using leaf in I1 (E). When all floral are developed at I2-7, the unidirectional BIOLOGY development of floral organs manifests the direction of the dorsoventral axis two primers nested to the intron. The transcript from squ1 is unspliced, larger (380 bp) than the one from wild type (150 bp), and LjUbi is used as the control for DEVELOPMENTAL (K, lines with arrow). Star, degenerate of I2; triangle, bract primor- dium; F, floral meristem. (Scale bars, 50 ␮m.) (L–N) The shapes of dorsal, lateral, template quantity. (D) Unrooted phylogram of protein NJ analysis of TCP domain and ventral petals in the wild type at late developmental stages. The mature region suing MGA3.1 (24). Branches with bootstrap support (1,000 replicates) Ն dorsal petal processes blade (triangle) and claw (arrow), and the lateral and 50% are indicated for each branch. All of the TCPs genes are from Arabidopsis ventral petals have blades (triangles) and stipes (arrows). (Scale bars, 1.0 mm.) (25); CYC and DICH, Antirrhinum; LCYC, Linaria vulgaris; LjCYCs, L. japonicus; PCFs, rice; TB1, maize.

Results more slowly than those in the ventral region. During late floral Inflorescence and Floral Development in L. japonicus. L. japonicus development, three types of petals develop along the dorsoven- (Gifu B-129) (9) is a perennial herb. When starting reproductive tral axis, and a key morphological landmark is the differentiation growth, each shoot becomes a primary inflorescence (I1), and produces a secondary inflorescence meristem (I ) at each node. of the vascular veins in petals. The various shapes of different 2 petals are distinctive (Fig. 1L) when the primary veins initiate in Floral meristems are generated from I2. We divided I2 devel- all petals. Then, the characteristic shapes of dorsal, lateral, and opment into seven stages, from I2-1 to I2-7 (Fig. 1 E–K). One I2 meristem initiates from the axil of the compound leaf at each ventral petals become visible (Fig. 1M) when the initiation of minor veins and freely ending veinlets begins and the differen- node (I2-1 and I2-2, Fig. 1 E and F). Bract primordia initiate at I2-3 (Fig. 1G) and become visible at I2-4 when the meristem of tiation of epidermal cell types commences. Further elaboration I2 ceases activity and becomes compressed (Fig. 1H). Then, of petal structures continues, giving rise to mature petals with normally three floral meristems are partitioned sequentially blades and claws in dorsal petals and blades and stipes in lateral from I2 (Fig. 1 I and J) and finally I2 differentiates, becoming and ventral petals (Fig. 1N), whereas various representative cell covered by trichomes (Fig. 1K). The development of floral types are generated in the epidermal layers of different petals dorsoventral asymmetry can be observed at the very beginning (see Fig. 4 H–J). of floral development when the floral organs initiate in a unidirectional order (23), and the dorsoventral axis becomes CYC Homologues in L. japonicus. Four TCP-box genes, LjCYC1, evident (Fig. 1K): organ primordia in the dorsal region develop LjCYC2, LjCYC3, and LjCYC5 (GenBank accession nos.

Feng et al. PNAS ͉ March 28, 2006 ͉ vol. 103 ͉ no. 13 ͉ 4971 Downloaded by guest on September 25, 2021 located in young leaves and at the base of I2 (data not shown). Therefore, we focus on LjCYC2, which was the most similar to CYC in expression pattern (Figs. 2A and 3 C–F). However, in contrast to the expression pattern of CYC, which was only observed in the dorsal region of floral meristem and dorsal floral organs, the earliest expression of LjCYC2 was found at the margin between I1 and I2 (Fig. 3A), and also became detectable in the center of the I2 meristem at I2-4, when it began to differentiate (Figs. 1H and 3B). Later, LjCYC2 was expressed in the dorsal region of floral meristems (Fig. 3 C and Fig. 3. Expression pattern of LjCYC2.(A–F) Longitudinal and transverse D), and then persisted in the dorsal organs of developing sections through the main apex of wild type were hybridized with LjCYC2 flowers, including the dorsal sepal, petal and stamen primordia antisense RNA probes labeled with digoxigenin-UTP. The transcript-specific (Fig. 3 E and F). The asymmetrical expression pattern of hybridization signal is visualized in blue. (G and H), Ectopic expression of LjCYC2 in the inflorescence (G) and floral meristems (H) in 35S::LjCYC2 trans- LjCYC2 in the floral meristem indicates that it could play a genic line SH3011. D(or V)Se, dorsal (or ventral) sepal primordium; D(or V)P, similar role to CYC in the control of floral dorsoventral dorsal (or ventral) petal primordium; D(or V)St, dorsal (or ventral) stamen; C, development in L. japonicus, whereas its expression in the carpel primordium. (Scale bars, 50 ␮m.) developing inflorescence meristem suggests a multifunction role during floral development.

DQ202475, DQ202476, DQ202477, and DQ202478), were Effects of Altered Expression of LjCYC2 in Transgenic L. japonicus. To isolated from L. japonicus. Phylogenetic analysis placed three test its function, the effects of reduced or constitutive expression of them, LjCYC1, LjCYC2, and LjCYC3, in a close clade of LjCYC2 were investigated in transgenic plants (Fig. 5 and including CYC and DICH (Fig. 2D), which correspond to the Supporting Text, which is published as supporting information on LEGCYC I(LjCYC1 and LjCYC2), and LEGCYC II (LjCYC3) the PNAS web site). All transgenic plants expressing LjCYC2 genes, respectively (21, 22). RNA in situ hybridizations were constitutively showed a specific effect in the lateral and ventral conducted to analyze their expression patterns. It was found petals. They displayed abnormal petal shapes and cell types in that LjCYC1 was expressed in I1 and dorsal regions of floral the adaxial epidermal layer to varying extents: the lateral petals primordia. Expression of LjCYC3 could not be detected by became more symmetrical and more like dorsal petals, possess- RNA in situ hybridization, and transcripts of LjCYC5 were ing dorsal-like conical cells mixed with jigsaw puzzle-shaped; the

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 typical transgenic flower of sense LjCYC2 transgenics. Arrow indicates the more symmetrical lateral petal. (C) A typical flower of antisense LjCYC2 transgenics. Star indicates 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 the keeled-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 indicates the 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). Arrows indicate 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 epidermal cells 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 and wax-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 shaped cells 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 of both 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 with ventral 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 ␮minH–V and 50 ␮minW–Y.)

4972 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600681103 Feng et al. Downloaded by guest on September 25, 2021 (Fig. 5C), although the variable phenotypes were weakened and vanished in succeeding generations. These data indicate that the precise expression pattern of LjCYC2 is important for floral SEE COMMENTARY development, suggesting other roles for LjCYC2, which could be distinct 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 Ϸ50,000 M2 families) to screen for mutations with abnormal floral symmetry. Several single recessive mutants with abnormal petal shape were obtained. One mutant was named squared standard 1 for the abnormal shape of the dorsal petal (Fig. 4D). squ1 seems to specifically affect the development of dorsal petal without other notable phenotypes, and its effect on petal de- velopment begins during initiation of the primary vein in the petal (data not shown). Genetic analysis showed that squ1 co-segregated with a molecular marker for the LjCYC2 locus in chromosome 2. Sequencing data confirmed that squ1 carried a point mutation at the splicing site of the intron in LjCYC2 (Fig. 2B), which gave rise to an abnormal transcript with lower expression level (Fig. 2C). squ1 has the capacity to encode a protein, in which the seventeen C-terminal amino acids are replaced by five others (Fig. 2C). This finding suggests that squ1 is a mutant allele of LjCYC2. Apart from its phenotype on the shape of dorsal petals, squ1 also has a specific effect on the epidermal cell shape in the dorsal petal. The cells do not all have the typical conical morphology, but include jigsaw puzzle-shaped cells characteristic of the lateral petals in the wild type (Fig. 4 N–P), indicating a correlation between the abnormal shape of squ1 dorsal petal and the reduction of dorsal identity. Therefore, the mutant phenotype of ljcyc2 is in agreement with transgenic Fig. 5. Comparison of primary inflorescence and secondary inflorescence (I2) data, confirming that LjCYC2 has a specific function in the between wild type and transgenic plants of L. japonicus.(A) In wild type, I2 control of dorsal identity. However, squ1 did not display any develops from the axil of the compound leaf at each node (arrows) and usually other detectable phenotype apart from the abnormal dorsal produces two flowers with diadelphous stamens (triangle). An internode is petal, in contrast to the range of phenotypes in transgenic plants indicated with a star. (B) In sense transgenic line SH3011, a few abnormal nodes with shorter internodes (stars) are normally generated before one or a with either sense or antisense transgene of LjCYC2. few I2 (arrows) replace I1. All stamens are separate from each other in the strong phenotype flowers with additional bract leaflets are generated. (C) Isolation of the kew1 Mutant. Another mutation only affects the Two I2 (indicated with two arrows) from the same node are occasionally appearance of the lateral petal and is named keeled wing in Lotus observed (star) and two flowers are frequently fused in various extents at 1 (kew1) for its similar phenotype to the mutant keeled wing (k) antisense transgenic plants (line SH3001). St, stamen; Br, bract; Fu, fusion in pea (24, 25), whose flowers bear the abnormal lateral petals flowers. (Scale bar, 1.0 cm.) (D) Expression levels of LjCYC2 in the wild-type and with the shape mimicking keel petals. In kew1, the lateral petals transgenic lines. The transcription of LjCYC2 cannot be detected in the leaves resemble the ventral in both petal shape and epidermal cell types BIOLOGY of wild-type and antisense transgenic lines (SH3001 and SH3005), and it is (Fig. 4 E and Q–S), but the unidirectional initiation of floral DEVELOPMENTAL reduced in floral tips of antisense transgenic lines, but accumulated at high level in both leaves and floral tips of the sense transgenic line (SH3011 and organ primordial (23), as well as the early development of lateral SH3012). NDT, not detected; #, standard deviation. petals up to the stage when the primary veins are initiated (Fig. 1L), are the same as the wild type (data not shown). This finding suggests that Kew1 is a specific factor in the control of lateral ventral petals were also more symmetrical, displaying patches of petal identity and functions at a late stage during petal devel- abnormal conical cells, whereas the dorsal petal maintained the opment. Our mapping experiment positioned kew1 in the short wild-ype shape and cell type (Fig. 4 B and H–J). This finding arm of chromosome 5, where it shares a macro synteny with the indicates that the ectopic expression of LjCYC2 can confer a region containing k in pea (data not shown), suggesting that kew1 dorsalizing effect. This effect was greatly enhanced in the GFP and k could be the same genetic locus controlling lateral petal tagged LjCYC2 line (SH0578), with all of the petals fully identity in papilionoid legumes. dorsalized and the normal dorsoventral asymmetry abolished (Fig. 4 G and W–Y). On the other hand, the antisense transgenic Interaction Between LjCYC2 and Kew1. We tested for interaction between LjCYC2 and Kew1 by crossing squ1 with kew1, because plants often displayed a phenotype with abnormal lateral petals mutants of both genes displayed effects on the shape and identity which resembled the ventral in both petal shape (Fig. 4C) and of dorsal and lateral petals, respectively. The squ1kew1 double epidermal cell type (data not shown), suggesting that reduced mutant, identified in the F2 population, had enhanced ventral LjCYC2 activity leads ventralization. Therefore, the dorsalizing phenotypes on dorsal and lateral petals, leading to all petals effect of LjCYC2 on petal identity was revealed in both sense and becoming ventralized to a different extent: the dorsal petal was antisense transgenic plants. However, altered LjCYC2 activity much smaller and did not expand outward (Fig. 4F), with some also gave rise to other morphological alterations, such as altered adaxial epidermal cells possessing the characters of normal inflorescence and floral structures (Fig. 5 B and C). In sense ventral petals (Fig. 4T); the lateral petals were ventralized but transgenic lines, I1 terminated with shortened internodes was larger than in kew1 (Fig. 4F), and the cell size and morphology observed frequently (Fig. 5B), and in primary antisense trans- were also ventralized (Fig. 4 R and U). Thus, LjCYC2 and Kew1 genic plants, two flowers were frequently fused to various extents have roles which are not limited to only the dorsal or lateral

Feng et al. PNAS ͉ March 28, 2006 ͉ vol. 103 ͉ no. 13 ͉ 4973 Downloaded by guest on September 25, 2021 petals, respectively, indicating that both genes are used together Papilionoideae. Caesalpinioideae is a paraphyletic assemblage from in the dorsoventral mechanism to determine petal identities. On which the monophyletic Mimosoideae and Papilionoideae arose. the other hand, there is no defect in floral organ initiation or Caesalpinioideae flowers are variable in morphology, although inflorescence development in the squ1kew1 double mutant (data often subtly zygomorphic, whereas Mimosoideae has mostly species not shown), suggesting that both LjCYC2 and Kew1 could have with actinomorphic flowers, and most species in Papilionoideae specific role in the control of petal identity along dorsoventral display prominent zygomorphic ‘‘pea’’ flowers. It has been shown axis. that the CYC-like genes have undergone two duplication events: an early one giving rise to LEGCYC I and LEGCYC II genes could Discussion have occurred before the evolution of Papilionoideae, and a later In this study, we exploited a model legume, L. japonicus,to one resulting in LEGCYC IA and LEGCYC IB have occurred conduct a robust functional analysis of a CYC-like gene, LjCYC2, during the early diversification of Papilionoideae (21, 22). So far, during zygomorphic floral development. We have demonstrated only LEGCYC IB genes, such as LjCYC2 in L. japonicus and its that LjCYC2, similar to CYC in A. majus, has an asymmetric homologue in pea (unpublished data), have been found to possess expression pattern in floral meristems and a dorsalizing activity dorsalizing activity and control the development of dorsal petals. In on petal identity, even though the zygomorphy of Leguminosae contrast to the expression pattern of CYC, LjCYC2 in L. japonicus is believed to have evolved separately from the Lamiales (1, 6, 7, is expressed transiently in the inflorescences (Fig. 3 A–F), and 13). A recent study has shown that the expression pattern of an altered LjCYC2 expression in transgenic plants had effects on both LjCYC2 orthologue is altered in the legume Cadia, which is the development of inflorescences and flowers (Fig. 5C), suggesting typical of a series of unrelated papilionoid genera with unusual, multiple developmental roles of LjCYC2 that are not shared with more or less radially symmetrical floral morphologies, support- CYC in A. majus. Although a mutant of LjCYC2 (squ1) affected ing a role for CYC homologues in the evolution of floral novelty only the identity of dorsal petals (Fig. 4D), it is possible that squ1 in legumes (H. L. Citerne, R. T. Pennington, and Q. C. B. Cronk, is a weak mutation giving rise to a partially functional protein, personal communication). Thus, these data reveal that CYC because its transcript is detectable and capable of encoding a homologues have been recruited in Leguminosae and play an mutant protein with the TCP and R domains intact. Alternatively, important role in the development of dorsoventral asymmetry. the function of LjCYC2 in the inflorescences may be redundant, It may not be coincidence that both LjCYC2 and CYC confer because the LEGCYC IA gene LjCYC1 is also expressed transiently dorsal activities in the two distantly related lineages. Although the in inflorescence meristems before it is asymmetrically expressed in precise functions of CYC and LjCYC2 on the development of the dorsal region of floral meristem. It has been shown that petal zygomorphy need to be clarified further, mutants of both genes asymmetry in A. majus could arise through a series of steps in which display similar effects on petals at the cellular level, such as the expression patterns of CYC and its homologue DICH are determining the epidermal cell types, size, and shape. This finding progressively elaborated and maintained from early floral meristem is in agreement with the general role of the TCP-box genes, which initiation through late stages of development, which should poten- is linked to the regulation of cell division and proliferation (26–28). tially reflect a sequence of evolutionary events (12). Similarly, the It suggests that a conserved function shared by TCP-box genes is dynamic expression pattern of LjCYC2 and LjCYC1 in the inflo- important for the dorsal activity of both LjCYC2 and CYC.Inthe rescences might be necessary for elaborating the dorsoventral axis Rosid Arabidopsis, which has radially symmetric flowers, the closest in floral meristems, and register the evolutionary course of their homologue to CYC and LjCYC2, TCP1, displays an asymmetric ancestor in Leguminosae. It is possible that the duplication of expression pattern transiently in the dorsal domain of floral mer- CYC-like genes and their functional divergence are generally im- istems, as well as in the adaxial region of axillary shoot meristems portant for the inherent dorsalizing activity of their ancestor to be (29). It has been speculated that an ancestral asymmetry had been elaborated and responded, so that the dorsoventral axis has been created in a common ancestor of A. majus and Arabidopsis by a evolved to produce the prominent zygomorphic flowers in different CYC͞TCP1-related gene, which presumably had the radially sym- lineages. Further analysis of the expression pattern and function of metrical flower, and further evolution of this gene, such as changes different CYC-like genes should provide more information about in timing or levels of expression and interactions with the target how gene duplication could contribute to the elaboration of dor- genes, may underlie repeated and independent recruitment of soventral axis and the development of zygomorphy among angio- CYC-like genes to control of zygomorphic floral development (29, sperms. 30). Consistently, our finding about the dorsalizing function of A genetic factor, Kew1 in L. japonicus, was identified in this study, LjCYC2 indicates that the similar evolutionary course of CYC-like the mutant of which affects the development of lateral petal only, genes have occurred independently in Leguminosae. Thus, the resulting in the lateral petal resembling the ventral in both petal CYC-like genes could help to define the dorsoventral axis through- shape and epidermal cell types (Fig. 4 E and Q–S). Because only the out angiosperms and there could be a common molecular origin for identity of lateral petals is altered and no other defect has been the mechanisms controlling floral zygomorphy. However, although found in the mutant kew1, Kew1 should have a specific role in the both CYC and LjCYC2 possess dorsalizing activity, they bring about control of lateral petal identity. Although their relationship needs the distinct zygomorphic flowers in A. majus and L. japonicus, further investigation, the similar phenotypes of the mutant kew1 respectively, which produce different internal symmetry in their and k suggest that Kew1 and K might be the same genetic compo- counterpart dorsal and ventral petals (Fig. 1 B and D), suggesting nent in the same pathway, and represent a lateralizing factor. We that LjCYC2 and CYC could have been integrated into different found that LjCYC2 could interact with Kew1, and the double genetic networks with divergent roles controlling the zygomorphic mutant bore ventralized petals. The enhanced phenotype of development. It is likely that the evolution of dorsoventral axis to squ1kew1 double mutant in the dorsal petal indicates that the control of zygomorphic flowers does not only depend on the extent activity of Kew1 could also be needed for the development of dorsal to which the ancestral dorsalizing activity has been elaborated, but petals, suggesting that there is a link between the dorsalizing and also rely on the way how it was responded in each zygomorphic lateralizing activities which should be important for the elaboration clade. Thus, the dorsalizing activity should be a primary and of dorsoventral axis. This possibility should be properly addressed synapomorphic function being recruited independently into the with the cloning of Kew1 and K from L. japonicus and pea in the mechanism to control the dorsoventral axis in different lineages. near future. There are some hints to how the dorsoventral axis might have Both single mutant, kew1 and squ1, and the double mutant evolved in the distantly related species independently. Leguminosae display defects on the development of dorsal and͞or lateral petals, consists of three subfamilies, Caesalpinioideae, Mimosoideae, and and their malfunction appears in the late stages of floral develop-

4974 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600681103 Feng et al. Downloaded by guest on September 25, 2021 ment when primary veins are initiated in petals (Fig. 1M). It has (SL1716 and SL1717) was used to detect the splicing in both wild been reported that morphologic differentiation of floral organs type and mutant. along the dorsoventral axis in papilionoid legumes occurs in the late SEE COMMENTARY stages of floral ontogeny (31). The specific activities and interaction Phylogenetic Analysis. Protein distance analysis was carried out by of LjCYC2 and Kew1 to control the identities of dorsal and lateral using MEGA version 3.1 (33) by the full length of protein petals in the late stage of floral development could provide the sequences from GenBank according to refs. 11, 12, 14, 18, 29, molecular basis for the characteristic floral ontogeny in legumes. and 34. Distance matrices used the PAM-Dayhoff model of On the other hand, the development of dorsoventral axis is evident amino acid substitution; the phylogenetic tree was constructed from the beginning of floral organ initiation, suggesting that there with the neighbor-joining method, and bootstrap analyses used could be a heterochronic role for the dorsalizing and lateralizing 1,000 resampling replicates. To simplify the analysis, only the activities in the control the development of dorsoventral axis and full-length TCP protein sequences of some representative and the petal identity respectively. In A. majus,thecyc mutant displays particular interest TCP-box genes were used. partial, and the cycdich double mutant complete, loss of dorsalized petals, with an increased number of dorsal organs, suggesting that Microscopy. Tissue for in situ hybridization was fixed overnight in CYC͞DICH have an early function to determine the floral organ 4% (wt͞vol) paraformaldehyde in phosphate buffer, pH 7.0, and numbers (11, 12). In contrast, although all petals are ventralized to embedded in Paraplast (Sigma). Nonradioactive in situ hybrid- a different extent, there is no increase of floral organ numbers in ization was performed essentially as described (35). Scanning the squ1kew1 double mutant, indicating that the LjCYC2 does not electron microscopy was performed on plastic replicas as de- play the same role as the one of CYC in the control of floral organ scribed earlier (36). Contrast and color balance were adjusted by initiation. These data further suggest that the independent recruit- using PHOTOSHOP 8.0 (Adobe, Mountain View, CA). ment of CYC-like genes within each zygomorphic clade might result in the acquisition of different functions, which could in turn have Transgenic Plants. Transformation of L. japonicus was carried out implication for the divergence of the mechanisms and account for as described (9). Details of transgenic constructions and trans- the distinct zygomorphy between different lineages. genic lines are described in Supporting Text. The alteration of LjCYC2 expression level in some lines were confirmed by Materials and Methods real-time PCR (Supporting Text). Plant Material and Growth Conditions. L. japonicus ecotypes Gifu B129 and Miyakojima (MG-20) were used in this study. All Mapping. kew1 was mapped by using an F2 mapping population, plants were grown at 20–22°C with a 16-h light͞8-h dark resulting from a cross between kew1 (from the Gifu B129 Ϫ Ϫ photoperiod at 150 mE⅐m 2⅐s 1. background) and Miyakojima (MG-20). In total, 6,000 F2 indi- Two mutants, kew1 and squ1 were isolated from a ␥ ray viduals were analyzed with markers flanking kew1. squ1 was mutagenized M2 population in the Gifu B129 background. After mapped with a F2 mapping population whose size was 160 plants. several backcrosses, in which 3:1 ratios of the wild type and Two primers (LJSSR0527 and LJSSR0528) were used as the SSR mutant were observed in the segregating populations, line markers for the genomic region containing LjCYC2. SH0389 for kew1 and line SH0120 for squ1 were established. We thank A Hudson, C. Kidner, R. T. Pennington, Q. C. Cronk, Gene Cloning and RT-PCR. Fragments of CYC-like genes were D. Bradley, M. Ambrose, P. Cubas, and E. Coen for their help and critical L. japonicus comments on the manuscripts; two anonymous reviewers for providing amplified from by using degenerate oligonucleotide help and advice; and Z. Xu, J. Li, Y. Xue, G. Wang, Y. Tian, H. Lin, Y. primers Pcyc1 and rPcyc2 (sequences of all primers are given in Wang, and S. Ge for their genuine encouragement and support for this Supporting Text)in5Ј-RACE using the 5Ј-RACE system experiment. 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