Evolution of novel floral organ identity in Aquilegia

Researchers: Elena Kramer, Claire Levy, Lynn Holappa, Bharti Sharma (collaborator) 

Aquilegia Model Organ Figure 1

A. An Aquilegia flower with select sepals, petals and stamens removed to reveal all internal organs. B. An Aquilegia flower with all sepals, petals, and stamens removed to reveal the staminodial whorl surrounding the carpels. C. Phylogenetic relationships among close relatives of Aquilegia with flower diagrams of key taxa. The evolutionary origins of key features are indicated by colored bars. D. The standard ABC model. E. A modified ABC model for Aquilegia. In all panels, sep = sepals, pet = petals, sta = stamens, std = staminodia, car = carpels.

    One of the main attractions to working with Aquilegia is its novel floral morphology (Fig. 1 above). The perianth is composed of two whorls of morphologically distinct petaloid organs: the first whorl petaloid sepals are ovate and flat and the second whorl petals are characterized by large nectar spurs (Fig. 1A). The 4-7 whorls of stamens have typical dicot morphology but internal to the stamens is one whorl of sterile organs termed staminodia, which are composed of a central filament flanked by lateral wings (Fig. 1B). These organs are clearly dissimilar from the outer stamens or inner carpels, and intermediates between the organ types are not commonly observed. While the ecological importance of the nectar spur is well established (Hodges 1997; Whittall and Hodges, 2007), the function of the staminodia is not well understood. It has been hypothesized, however, that they play a role in protecting the young ovaries from herbivory damage (Voelckel et al., 2010) or possibly fungal infection. The close phylogenetic relatives of Aquilegia put the evolution of these novel traits in context (Fig. 1C). The sister genus Semiaquilegia has a nectary pocket but no spur and its inner stamens can be reduced to irregular, sterile filaments that are not laterally expanded (Fig. 1C; Tucker and Hodges 2005). Urophysa is the next outgroup genus and it shows an even weaker differentiation of the nectary pocket and occasional sterile inner staminodes (Wang and Cheng 2007).

Aquilegia Figure 2

Fig. 2. Summarized expression patterns of three AP3 parlous and the single PI homolog.

    Understanding the genetic basis of Aquilegia’s distinct morphology began with a full characterization of B gene homologs (Fig. 2 below; Kramer et al., 2007). There are three paralogs of AP3, termed AqAP3-1, -2 and -3, and one PI homolog, AqPI. The AqAP3-1 expression domain is broad at early developmental stages but quickly becomes stronger in the staminodia, suggesting that it may play an important role in promoting staminodial identity but could also contribute to petal or stamen identity. AqAP3-2 is primarily expressed in stamens but also in later petal development. In contrast, AqAP3-3 expression is petal-specific throughout development. AqPI is likely to function with all of these paralogs, both based on its broad expression and the fact that AqPI heterodimerizes with all three AP3s. Lastly, at very late stages, AqPI and AqAP3-1 and -2 are detected by RT-PCR in the sepals, raising the possibility that they contribute to petaloidy of the sepals. We have now used RNAi-based VIGS (Gould and Kramer 2007) with the tobacco rattle virus platform to obtain specific silencing, confirmed by qRT-PCR, of AqPI, AqAP3-1, -2 and -3 as well as dual silencing of AqAP3-1/2.

Silencing of AqPI in A. vulgaris produced a strong B class phenotype of petal to sepal and stamen to carpel transformation (Kramer et al. 2007). In addition, the novel staminodia were transformed into carpels, confirming that these organs are controlled by the Aquilegia B gene homologs. The strong transformations observed in these plants were expected since all three AP3 homologs appear to function as heterodimers with AqPI. Silencing of AqAP3-3 alone results in transformation of petals into sepals with no effect on the development of the other floral organs (Sharma et al. 2011). Conversely, silencing of AqAP3-1 alone causes partial transformation of staminodia into carpels while silencing of AqAP3-2 results in severely stunted stamens (Sharma and Kramer 2013). Only when both AqAP3-1 and -2 are silenced together are the stamens completely transformed towards carpel identity. These results suggest an ABC model for Aquilegia in which ancient duplications in the AP3 lineage have been more recently utilized to evolve a new organ identity, which resulted in neofunctionalization of AqAP3-1 (Fig. 1E) In addition, we are studying the roles of the Aquilegia AGAMOUS paralogs AqAG1 and AqAG2 in establishing staminodium identity.

Beyond this candidate gene approach, we plan to examine 1) the evolution of the novel staminodium organ identity program (see below), and 2) the developmental and genetic basis of nectar spur evolution (see the Petal Spurs page).

    In order to move beyond our characterization of floral organ identity homologs, we have used the PALM Laser Microbeam to isolate multiple developmental stages of stamen and staminodia primordia from early stage Aquilegia floral meristems. A total of 1 million m2 of tissue was microdissected for each of three replicates of five sample classes (Fig. 3 below): stamen primordia at inception before staminodium initiation (class st0); separate stamen and staminodium primordia immediately after carpel initiation (classes st1 and sd1), at which stage staminodia can be unequivocally identified; and separate stamen filaments and staminodia (classes st2 and sd2). We chose to use only stamen filaments for class st2 because the anthers express a high number of genes related to microsporogenesis, which we already know will be differentially expressed (Voelckel et al, 2010). Also, rare stamen/staminodium chimeras consist of a complete staminodium with a small terminal filament and aborted anther, suggesting that the entire staminodium corresponds to the stamen filament rather than any part of the anther. All samples were sequenced on an Illumina Genome Analyzer II. This RNA-seq dataset has been analyzed to identify candidate genes for both the upstream regulation of AqAP3-1 and -2, and the potential downstream targets of these proteins. In particular, we are interested in the pathways that may be responsible for delimiting the staminodium whorl from the rest of the stamens, that promote lateral outgrowth of the staminodia but limit expansion in the stamen filament, and that prevent post-anthesis abscission of the staminodium but promote it in the stamen.

To complement this molecular genetic approach, Claire Levy and her undergrad mentee Kate Freedberg have been studying the late developmental patterns in staminodia, which exhibit an extremely late connate fusion along their lateral margins. Claire and Kate are also collaborating with Scott Hodges to conduct the first tests of whether staminodia contribute to fitness in the field.

Gould, B. and Kramer, E. M. (2007) Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Aquilegia (columbine, Ranunculaceae). Plant Methods, 3:6.

Hodges, S.A. (1997) Rapid radiation due to a key innovation in columbines., in Molecular evolution and adaptive radiation. T.J. Givnish and K.J. Sytsma, Eds. Cambridge University Press: Cambridge. p. 391-405.

Kramer, E. M., Holappa, L., Gould, B., Jaramillo, M. A., Setnikov, D. and Santiago, P. (2007) Elaboration of B gene function to include the identity of novel floral organs in the lower eudicot Aquilegia (Ranunculaceae). Plant Cell, 19:750-766.

Sharma, B., Guo, C., Kong, H., and Kramer, E. M. (2011) Petal-specific subfunctionalization of an APETALA3 paralog in the Ranunculales and its implications for petal evolution. New Phytologist, 190:870-883.

Sharma, B. and Kramer, E. M. (2013) Sub- and neofunctionalization of APETALA3 paralogs have contributed to the evolution of novel floral organ identity in Aquilegia (columbine, Ranunculaceae). New Phytologist, 197:949-957.

Tucker, S.C. and Hodges, S. A. (2005). Floral ontogeny of Aquilegia, Semiaquilegia, and Isopyrum (Ranunculaceae). Int’l J Plant Sci, 166:557-574.

Voelckel, C., Borevitz, J., Kramer, E. M., and Hodges, S. A. (2010) Within and between whorls: comparative transcriptional profiling of Aquilegia and Arabidopsis. PLoS ONE, 5:e9735.

Whittall, J.B. and Hodges, S.A. (2007) Pollinator shifts drive increasingly long nectar spurs in columbine flowers. Nature, 447:706-710.