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A central goal of evolutionary developmental biology is to investigate the relationship between the evolution of developmental control genes and phenotypic variation. More than a century after Charles Darwin referred to the origin and rapid radiation of the angiosperms as an ‘abominable mystery’ (Darwin & Seward, 1903), there are still many unanswered questions about the underlying causes of this phenotypic variation. The flower, and the interactions with pollinators that it enhances, is considered one of the most important innovations that have allowed angiosperms to become the dominant element of today's flora (Crepet, 1984). Studies of closely related species whose flowers vary in their pollination syndromes are instrumental in understanding the evolution of the associated adaptive traits.
Since the sequencing of complete genomes from a few model plants, many genes involved in different aspects of floral organ identity and differentiation have been characterized. This knowledge allows for a more informed candidate-gene approach towards understanding adaptive phenotypic diversity throughout the angiosperm phylogeny. Extending our understanding of the role of developmental control genes to taxa that are particularly informative phylogenetically is an important step towards understanding the evolution of floral diversity.
As part of a broader quest to investigate the molecular basis of morphological differences between flowers of related species, we set out to study variation that has evolved as a result of adaptation to different pollination agents: wind or insects. Our work took a comparative approach to the investigation of the expression and function of a MYB family transcription factor, a candidate gene for determining features of the floral epidermis that affect pollination in species of the non core eudicot genus Thalictrum.
Thalictrum comprises c. 190 species of herbaceous perennials distributed in temperate regions worldwide (Tamura, 1993) with 22 species in North America (Trelease, 1886; Park & Festerling, 1997). It belongs to the family Ranunculaceae, sister to a clade containing Aquilegia, the columbines (Hoot, 1995). Thalictrum provides a spectrum of variation in floral traits coupled with two pollination mechanisms (reviewed in Pellmyr, 1995) and four breeding systems.
Thalictrum flowers are apetalous, that is, they completely lack petals; however, different species show varying degrees of petaloidy (characters that are usually associated with petals, such as color and conical cells) in sepals or stamens. Ongoing phylogenetic reconstruction and character mapping suggest that insect pollination is the ancestral condition in the genus; wind pollination evolved early followed by the evolution of unisexual breeding systems such as andromonoecy and dioecy (J. Brunet, V. S. Di Stilio & A. Liston, unpublished).
In this study, we compare three representative species: Thalictrum thalictroides has the ancestral features of insect pollination syndrome (Kaplan & Mulcahy, 1971), consisting of expanded, ‘showy’ sepals that are white or purple and contain conical cells in the adaxial epidermis (Figs 1a–c, 2a–d); closely related Thalictrum filamentosum is a representative of a different type of insect pollination syndrome, consisting of petaloid stamens containing conical cells and/or pigments (Figs 1d–f, 2e–h) and Thalictrum dioicum, with derived wind pollination features (Kaplan & Mulcahy, 1971) of inconspicuous, small, green flowers without conical cells, with extended stigmatic surfaces, trichomes, many pendulous stamens, and often dioecious or monoecious (Figs 1g–i, 2i–l).
Figure 1. Floral diversity in the genus Thalictrum (Ranunculaceae), representative species. (a–c) Species with petaloid sepals, mostly insect pollinated. (a) Thalictrum delavayi; (b) Thalictrum rochebrunianum;(c) Thalictrum uchiyamai. (d–f) Species with petaloid stamens, mostly insect pollinated. (d) Thalictrum kiusianum; (e) Thalictrum punctatum; (f) Thalictrum aquilegifolium (with pink stamen filaments). (g–i) Species with wind-pollination syndrome (nonshowy, many dioecious). (g) Thalictrum arsenii; (h) Thalictrum fendleri (carpellate); (i) Thalictrum alpinum.
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Figure 2. Characterization of flowers and their epidermal cell types in three representative species of Thalictrum used in this study. (a–d) Thalictrum thalictroides. (a) Showy insect-pollinated hermaphroditic flowers; (b) detail of petaloid sepal; (c) Scanning electron microscopy (SEM) of adaxial sepal epidermis showing conical-papillate cells; (d) transverse section of fresh sepal showing conical cells on adaxial surface. (e–h) Thalictrum filamentosum. (e) Showy hermaphroditic flower; (f) detail of stamen with expanded petaloid filament; (g) SEM of stamen filament epidermis showing slightly conical cells; (h) transverse section through stamen filament showing conical cells. (i–l) Thalictrum dioicum. (i,j) Green, small and inconspicuous wind pollinated staminate and pistillate flowers of this dioecious species; (k) SEM of adaxial sepal showing flat epidermal cells; (l) transverse section of sepal showing flat cells on adaxial surface. Bars, (a,b,e,f,i,j) 1 mm, (c,d,g,h,k,l) 50 µm. Ad, adaxial; Ab, abaxial.
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The presence of conical cells in the adaxial petal epidermis of most angiosperms is an important component of petal appearance that enhances color and brightness (Noda et al., 1994). In Antirrhinum majus (snapdragon), the MYB transcription factor MIXTA and its relatives play a role in petal coloration by promoting the formation of conical epidermal cell shape (rather than by affecting pigment concentration), resulting in conical cells that affect light reflection, making them more attractive to insect pollinators (Noda et al., 1994; Glover & Martin, 1998). MIXTA and MIXTA-like are members of the R2R3-MYB family of transcription factors consisting of a conserved DNA-binding (MYB) domain comprised of two repeats (R2 and R3). A motif in the C-terminal domain characterizes them as members of Subgroup 9 (Kranz et al., 1998; Stracke et al., 2001).
Three other MIXTA relatives have been identified in snapdragon, known as AmMYBMIXTA-like (ML) 1–3, involved in aspects of epidermal cell fate that relate to adaptation to pollinators: conical cells that intensify petal color, trichomes that collect pollen, and mesophyll cell expansion that provides a grasping zone for pollinators (Martin et al., 2002; Perez-Rodriguez et al., 2005).
While MIXTA and MYBML1 have so far been found only in Antirrhinum, other MIXTA-like genes have been isolated from Petunia (PhMYB1; van Houwelingen et al., 1998), and Arabidopsis (Romero et al., 1998; Baumann et al., 2007). However, no related genes have yet been isolated or characterized functionally outside of the core eudicots. Functional characterization of PhMYB1 and AmMYBML2 showed that they promote directional cell expansion and have functions overlapping with that of MIXTA (Baumann et al., 2007). A detrimental effect of mutations in these genes on pollination visitation and fitness has been shown in the field for Antirrhinum (Glover & Martin, 1998) and has been implied, because of effects on flower shape and apparent size, in Petunia (Baumann et al., 2007).
Our goal is to identify a tractable floral adaptive trait, characterize the gene responsible for it and compare its expression and function across species representative of the different morphologies. To that end, our study takes the following approach: (1) identification of a micromorphological trait that correlates with petaloidy (conical cells), given that petals are absent but other organs take over the attraction role (sepals and stamens); (2) cloning of the gene responsible for making conical cells, taking a candidate gene approach (ThalictrumMYBML2); (3) comparative expression of the candidate gene among species with different placement of conical cells or lack thereof; and (4) characterization of gene function using a transgenic approach in a model plant system (tobacco).
Thalictrum provides an opportunity to study the genes underlying changes in floral morphologies associated with adaptation to different pollination agents. In this study, we have identified and characterized a gene encoding a floral transcription factor responsible for petaloid features associated with insect pollination. Importantly, this system also allows the dissection of organ identity from organ differentiation and function: in this case, a feature that is typical of petals (conical cells), is expressed ectopically in stamens and sepals, conferring on them the function (but not the identity) of petals.