An apparent reversal in floral symmetry in the legume Cadia is a homeotic transformation

He´ le` ne L. Citerne*†‡, R. Toby Pennington*, and Quentin C. B. Cronk§

*Royal Botanic Garden Edinburgh, 20a Inverleith Row, Edinburgh EH3 5LR, United Kingdom; †Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; and §University of British Columbia Botanical Garden, Room 206, Campbell Building, 6804 Southwest Marine Drive, Vancouver, BC, Canada V6T 1Z4

Edited by Michael J. Donoghue, Yale University, New Haven, CT, and approved June 26, 2006 (received for review February 8, 2006)

Within papilionoid legumes, characterized by flowers with strong bilateral symmetry, a derived condition within angiosperms, Cadia (Cadia purpurea) has reverted to radially symmetrical flowers. Here, we investigate the genetic basis of this morphological reversal. Two orthologues of the floral symmetry gene CYCLOIDEA (CYC) demarcate the adaxial (dorsal) region of the flower in typical papilionoid legumes. In the model legume Lotus japonicus, one of these LegCYC genes has been shown, like CYC, to be required for the establishment of floral bilateral symmetry. This study shows that these genes are expressed in the adaxial region of the typical papilionoid flower of Lupinus, which belongs to the same papil- ionoid subclade as Cadia.InCadia, these genes also are expressed, but the expression pattern of one of these has expanded from the adaxial to the lateral and abaxial regions of the corolla. This result suggests that the radial flowers of Cadia are dorsalized and, therefore, are not a true evolutionary reversal but an innovative homeotic transformation, where, in this case, all petals have acquired dorsal identity. This study raises a question over other putative reversals in animals and plants, which also may be cryptic innovations.

developmental gene ͉ floral evolution ͉ CYCLOIDEA ͉ Leguminosae Fig. 1. Mature whole and dissected flower of L. nanus and C. purpurea.(a) (Fabaceae) Detail of an L. nanus flower, showing strong bilateral symmetry typical of papilionoid flowers. Individual standard (D, dorsal), wing (L, lateral), and keel (V, ventral) petals are shown, as well as the monadelphous (with united ost members of the plant family Leguminosae (Fabaceae) filaments) androecium with stamens of different lengths (lateral view on the Mhave flowers that are characterized by a single axis of left, and dissected dorsal and lateral͞ventral stamens on the right; the three symmetry (bilateral symmetry or zygomorphy). The majority of dorsal stamens are shorter than the lateral and ventral ones). (b) C. purpurea legume species, from the subfamily Papilionoideae and account- flowers are radially symmetrical, with equal dorsal (D), lateral (L), and ventral ing for Ϸ5% of all flowering plant species, are characterized by (V) petals and stamens. having typical ‘‘pea’’ (papilionoid) flowers with pronounced bilateral symmetry particularly in the corolla (illustrated here by The early development of Cadia (Cadia purpurea) flowers is Lupinus nanus; Fig. 1a). The typical papilionoid flower has three similar to that of most papilionoid species with zygomorphic distinctive petal types: a single upper (adaxial or dorsal) petal flowers where sepals, petals, and stamens are initiated unidi- (the ‘‘standard’’), two lateral petals (‘‘wings’’), and two lower rectionally, starting on the abaxial side (6). In typical papilionoid (abaxial or ventral) petals (‘‘keel’’ petals). The androecium of legumes, although organogenesis is asymmetric, a phase of typical papilionoids also is bilaterally symmetrical, with stamens uniform organ growth precedes the unequal development along of different lengths along the dorsoventral axis, the adaxial the dorsoventral axis, and differential organ development occurs EVOLUTION stamens being shorter (Fig. 1a). However, within the Papilion- at an advanced stage of floral ontogeny (7). By contrast, in C. oideae, a number of unrelated genera have radially or nearly purpurea, floral organs continue to develop equally after orga- radially symmetrical flowers. The flowers of these atypical nogenesis until maturity (6). Tucker (6) therefore interpreted the species frequently have been considered primitive (reviewed in phenotype of C. purpurea as ‘‘neotenous,’’ that is, retaining the ref. 1), but more recent phylogenetic studies suggest that they are characteristics of early flower development (uniform growth) derived from a zygomorphic ancestral state (2, 3). An example and not entering the asymmetric phase. In genetic terms, this is Cadia Forsk., a genus of seven species of small shrubs from interpretation suggests that the radial phenotype of C. purpurea Arabia, Madagascar, and eastern Africa. Cadia has actinomor- may be caused by a loss of expression of genes that affect floral phic pendent flowers in solitary or few-flowered axillary racemes (ref. 4; Fig. 1b). These flowers produce abundant nectar but no scent, suggesting these may be pollinated by birds (1). Conflict of interest statement: No conflicts declared. Cadia occupies a relatively isolated phylogenetic position (J. S. This paper was submitted directly (Track II) to the PNAS office. Boatwright and D. Edwards, personal communications) within Data deposition: The sequences reported in the paper for Lupinus nanus LEGCYC1A and the genistoid clade of papilionoid legumes (83 genera and 2,350 LEGCYC1B and Cadia purpurea LEGCYC1A and LEGCYC1B have been deposited in the species; ref. 5). Its closest relatives are from the tribe Podalyrieae GenBank database (accession nos. AY382156, AY382155, AY225826, and AY225825). (J. S. Boatwright and D. Edwards, personal communications), ‡To whom correspondence should be addressed. E-mail: h࿝[email protected]. which have typical zygomorphic papilionoid flowers. © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600986103 PNAS ͉ August 8, 2006 ͉ vol. 103 ͉ no. 32 ͉ 12017–12020 Downloaded by guest on September 24, 2021 Fig. 3. RT-PCR analysis of LegCYC1A (1A) and LegCYC1B (1B) from whole- flower buds at early developmental stages (Ϸ1 mm diameter) and dissected floral tissue from L. nanus and C. purpurea flowers are later stages. The corolla and androecium in both species were dissected as described in Materials and Methods. Intron splicing in both genes allowed to distinguish between cDNA and genomic DNA. cDNA products amplified by actin-specific primers were used as a positive control. gDNA, genomic DNA; Ϫve, negative control (no template); D, dorsal; L, lateral; V, ventral. Fig. 2. Patterns of RNA in situ hybridization of LegCYC1A and LegCYC1B in L. nanus inflorescenses (a and b, respectively), with details of floral meristems at different development stages (d–f, LegCYC1A; g–i, LegCYC1B). Longitudi- side throughout floral development in another papilionoid le- nal sections of L. nanus inflorescences show floral meristems (fm) in the axil of gume, Lupinus (L. nanus), from the genistoid clade. L. nanus has bracts (B) on the abaxial side. More mature flowers are at the base of the inflorescence. RNA from LegCYC1A and LegCYC1B is detected in the adaxial typical zygomorphic pea flowers, suggesting that these genes are part of floral meristems before organogenesis (d and g) and during floral candidates for the control of floral symmetry in genistoid organ development (e, f, h, and i). Although both copies have a similar legumes. The expression of these two LegCYC genes is examined expression pattern, LegCYC1B appears to have a wider expression domain in the radially symmetrical flowers of Cadia (C. purpurea), which, than LegCYC1A. Negative control (sense probe) is shown (c). Ad, adaxial; Ab, like Lupinus, is a genistoid legume (2). Comparison of LegCYC abaxial; St, stamen; AbS, abaxial sepal. (Scale bars: a–c, 500 ␮m; d–h, 200 ␮m; expression pattern between an unusual legume such as C. f and i, 250 ␮m.) purpurea and a related zygomorphic species can illuminate the genetic basis of morphological reversal in relation to floral symmetry in legumes. The reversibility of evolution is an im- organ differentiation during the latter stage of development of portant problem, but few examples of apparent phenotypic typical zygomorphic papilionoid flowers. reversals have been investigated genetically (reviewed in ref. 13). Genes that are important for the control of floral symmetry Here, we establish whether the apparent reversal of floral have been identified in Antirrhinum majus L. (Veronicaceae, symmetry in Cadia is the result of loss of expression of candidate Lamiales) (8–10). Two closely related genes CYCLOIDEA genes, which could be described as ‘‘true reversal,’’ or is due to (CYC) and DICHOTOMA (DICH) demarcate the dorsal region modified gene expression causing the replication of ancestral of the flower and affect the development of floral organs along states. the dorsoventral axis. CYC and DICH expression is maintained throughout floral development in the adaxial region (8, 9). Results Double knockout mutants for CYC and DICH have a fully LegCYC1A and LegCYC1B RNA was detected in floral tissue of radially symmetric phenotype characterized by ventralization of L. nanus in a pattern similar to Antirrhinum CYC (ref. 8; Fig. 2). floral organs (i.e., resembling the ventral phenotype of wild-type Both genes were detected in floral meristems before organo- flowers) (8). genesis, on the adaxial side of the meristem (Fig. 2 d and g). At CYC͞DICH homologues have been identified in legumes, more advanced developmental stages, both genes were detected referred to as LegCYC (11). There is strong functional evidence in the corolla (Fig. 2 e, f, h, and i). Similar to CYC, expression that at least one LegCYC gene, LjCYC2 (Lotus japonicus 1 in ref. of LegCYC1B in the dorsal petal was found in the inner cell layers 11), is involved in the control of floral symmetry in the model at the site where cell division was repressed early in organogen- papilionoid legume Lo. japonicus (12), a member of the robin- esis. Although the expression domains of LegCYC1A and ioid clade. Like CYC, LjCYC2 is expressed in the adaxial region LegCYC1B are largely overlapping, suggesting functional redun- of developing flowers and has been found to be important for dancy, LegCYC1A appears to have a reduced expression domain establishing dorsal identity (12). Lotus plants expressing LjCYC2 relative to LegCYC1B. constitutively have lateral and ventral petals that are similar to RT-PCR results in L. nanus suggest that, in agreement with dorsal petals in shape and epidermal cell type, whereas those the findings in situ, both LegCYC1A and LegCYC1B are florally with reduced LjCYC2 activity have lateral petals with a ventral- expressed (Fig. 3) and only in the adaxial part of the developing ized phenotype (12). We show here that two LegCYC genes, flower (Fig. 3). RT-PCR also shows that this expression is found belonging to the same gene clade (LegCYC1) as LjCYC2 (11) in floral buds at more advanced developmental stages. Both (sequences given in Fig. 4, which is published as supporting copies are transcribed at this stage not only in the standard information on the PNAS web site) are expressed on the adaxial (dorsal) petal but also in the dorsal anthers.

12018 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600986103 Citerne et al. Downloaded by guest on September 24, 2021 In C. purpurea, RT-PCR from young flower buds revealed that change in cis regulation also has led to the expansion of the LegCYC1A and LegCYC1B also are expressed in young floral expression domain of LegCYC1B in C. purpurea. buds (Fig. 3). However, the pattern of expression of these genes The occurrence of a putative ancestral state such as radial in dissected tissue from the corolla and androecium at an symmetry within a clade that has a derived character (zygomor- advanced developmental stage are different from each other and phy) is frequently referred to as an evolutionary reversal. Our from their L. nanus orthologues. In the corolla of C. purpurea, results indicate that from a genetic point of view, the radial LegCYC1A is expressed only in the dorsal petal, and its level of symmetry of Cadia is an evolutionary innovation correlated, in expression appears moderate to weak (Fig. 3). LegCYC1B, part, by the expansion of the expression domain of a CYC-like however, is expressed in all petals (Fig. 3), showing an expansion gene in the corolla. The functional significance of this expression of the expression domain of this gene with respect to L. nanus, pattern is suggested by transgenic experiments in Lo. japonicus which is consistent with the radial phenotype of the corolla. In (12). Overexpression mutants showing an expansion of expres- addition, unlike in L. nanus, neither LegCYC1A nor LegCYC1B sion of LjCYC2 displayed a dorsalized phenotype (12), support- appear to be expressed in the androecium of C. purpurea (Fig. 3). ing the hypothesis that changes in LegCYC1B expression in Cadia have an effect on flower development. Because CYC is a marker Discussion for dorsal identity, these changes can be considered homeotic, This study shows that two CYC-like genes, based on their with the lateral and ventral petals of Cadia assuming a dorsal expression pattern in the zygomorphic flowers of the typical phenotype. This interpretation also is supported by morphology. papilionoid legume Lupinus, are likely to control floral symme- In Cadia, the five petals are large and bilaterally symmetric, try in the genistoid clade to which Lupinus belongs. These genes, features that are typical of the papilionoid standard petal. By which have duplicated independently from CYC and DICH (11), contrast, wing and keel petals in typical papilionoid flowers are may be functionally redundant. Therefore, not only do CYC-like asymmetric and small relative to the standard. Partial dorsal- genes appear to have been recruited independently for the ization also has been noted in Mohavea confertiflora, where control of zygomorphy in Lamiales and legumes (12), but stamen number is reduced from four to two compared with its maintenance of partially redundant copies also has occurred close relative A. majus and is correlated with expansion of CYC independently in these lineages. The role of these genes in the and DICH expression from the adaxial to the lateral region (14). development of zygomorphic flowers in this legume clade is Although loss of function is often perceived as the simplest supported by functional evidence from the orthologous LjCYC2 scenario to explain possible reversals to radial symmetry, it has in Lo. japonicus (12). This result provides a framework for been suggested that, on close morphological inspection, many examining the genetic control of floral symmetry in a clade that shifts zygomorphy to actinomorphy involve other genetic or contains atypical species with actinomorphic flowers. developmental changes (15). Our findings support the hypoth- esis that such homeotic-like transformations may play an im- In C. purpurea, like in Lupinus, both LegCYC1A and portant role in establishing morphological diversity. LegCYC1B are transcribed in early developing flowers. Taking ontogeny into account, this result is not surprising because early Materials and Methods development of C. purpurea flowers is similar to that of most RNA in Situ Hybridization. RNA in situ hybridization was carried papilionoid species with zygomorphic flowers, with unidirec- out as described by K. Barton (available upon request; The tional organ initiation starting on the abaxial side (6). It remains Carnegie Institute of Washington, Stanford, CA) on 7-␮m to be demonstrated whether LegCYC genes have the same longitudinal sections of L. nanus inflorescences fixed in 4% expression pattern in early floral development in C. purpurea paraformaldehyde. Digoxygenin-labeled probes specific to compared with typical papilionioids. During the latter stages of LegCYC1A and LegCYC1B were prepared from linearized PCR floral development, whereas floral organs undergo unequal fragments, amplified from the 5Ј region of the ORF by using growth along the dorsoventral axis in typical papilionoid legumes primers LEGCYC1-F2 (5Ј-CTT TCY TTA ACC CTG AAA such as Lupinus, they develop equally in C. purpurea (6). This ATG CTT C-3Ј) at the start of the ORF and LEGCYC1A-R1 equal development could suggest that genes that control dorso- (5Ј-CTA CYA CTA CCC CTT CTG G-3Ј)͞LEGCYC1B-R1 ventral asymmetry are not expressed after organogenesis in C. (5Ј-CAA GCS GGT TCC TTY TGT G-3Ј), respectively (prim- purpurea. The molecular data presented here, however, suggest ers and PCR conditions described in ref. 16), and cloned into that rather than failing to maintain CYC expression during the pCR4 plasmid (Invitrogen, Carlsbad, CA). later stages of flower development, one CYC-like gene, Extensive and exhaustive attempts to provide in situs of Cadia LegCYC1B, is expressed in all five petals of C. purpurea. Another flowers were unsuccessful. Fixation and sectioning were very copy, LegCYC1A, is expressed adaxially but may be down- problematic, probably because of the adaptation of the flowers regulated. The expansion of LgCYC1B expression appears to be to their xeric habitat (tough sepals covered in filamentous EVOLUTION whorl-specific. In the androecium, neither LegCYC1B or trichomes). LegCYC1A mRNA was detected in C. purpurea, although ex- pression was found in the dorsal region in L. nanus. The apparent RT-PCR. Tissue for RT-PCR was collected from young floral buds loss of CYC expression in the stamens is intriguing but difficult (Ϸ1 mm diameter). Tissue also was collected from the corolla to interpret. Mature stamens in C. purpurea are identical mor- and androecium of flowers at more advanced and comparable phologically, and the Cadia androecium is actinomorphic, but it developmental stages in L. nanus and C. purpurea, during the is hard to establish whether the stamens have a ventralized latter stages of organ enlargement where these had undergone phenotype. Without more information on the role of CYC in a high degree of dorsoventral differentiation in L. nanus and stamen development of other legume species, it is difficult to where the flowers, still in bud, could be easily dissected. Floral place our observations in context. orientation in C. purpurea was determined with respect to the The expression of LegCYC1B in the corolla is reminiscent of gynoecium, which is the only part of the flower showing dorso- the backpetals mutation in Antirrhinum (9). This mutant has ventral asymmetry. Total RNA was extracted by using Qiagen ectopic expression of CYC in the lateral and ventral petals. A RNeasy RNA extraction kit (Valencia, CA) from the following transposon insertion in an AT-rich site Ϸ4.2 Kb upstream of start tissues: the dorsal, lateral, and ventral petals in L. nanus, and codon is believed to affect a cis-acting region that normally each individual petal from a single flower in C. purpurea, the suppresses CYC transcription during the latter stages of devel- three adaxial stamens separated by a groove from the seven opment in wild-type Antirrhinum flowers (9). It may be that a lateral and ventral stamens (combined) in L. nanus, and the

Citerne et al. PNAS ͉ August 8, 2006 ͉ vol. 103 ͉ no. 32 ͉ 12019 Downloaded by guest on September 24, 2021 dorsal (three uppermost) lateral (two on either side) and ventral flowers. Actin was used as a positive control (using primers (three lowermost) stamens in C. purpurea. cDNA was synthe- described in ref. 17). To ensure that the quantity of PCR sized by using Qiagen Omniscript RT kit. RT-PCR was carried products was comparable between samples, actin products were out by using the locus-specific primers LEGCYC1A-F1 (5Ј- CCA assayed between 20 and 35 cycles. PCR products are shown in the GAA GGG GTA GTR GTA G-3Ј)͞LEGCYC1B-F1 (5Ј-CAC linear phase after 30 cycles. ARA AGG AAC CWG CTT G-3Ј) and primer LEGCYC1-R1 (5Ј-CAC TCY TCC CAR GAY TTT CC-3Ј), which is down- We thank E. Coen and D. Luo for making this project possible; M. stream of the short intron at the 3Ј end of the LEGCYC ORFs Hollingsworth, A. Clark, C. Baxter, and K. Coenen for technical (primers described in ref. 16). Locus specificity was confirmed by assistance; and the editor and two anonymous reviewers for constructive sequencing of RT-PCR products. RT-PCRs were repeated with comments on the manuscript. This work was supported by the Carnegie cDNA from separate RNA extractions of tissue from individual Trust for the Universities of Scotland.

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