The variety of life-from apples to zebras-is beautiful, engaging and excites the imagination. The question of how different forms arise is one of the most fascinating and important in all of biology. The flowering plants offer a wonderful opportunity to investigate the genetic basis evolutionary novelty, especially in the context of unique forms of floral organs. Most flowers display a standard set of organs-sepals, petals, stamens, and carpels-but some have evolved fifth organ types. This project uses the new model system Aquilegia (columbine) to understand the genetic basis of novel floral organ identity and elaboration. In columbines, standard floral organs may develop fantastic new features, such as petal nectar spurs that vary in length from a few mm to over 12 cm. The project builds on previous studies to understand i) how gene duplication has contributed to the evolution of a fifth, distinct form of floral organ, the staminodium; ii) what other genetic changes contributed to the evolution of the staminodium; and iii) how development of Aquilegia's petal nectar spurs is controlled from a hormonal and genetic standpoint. Training our next generation of scientists requires getting students excited about the scientific process and familiarizing them with the real day to day practice of science. Genetic annotation of the recently sequenced Aquilegia genome will be used as the basis for a lecture/lab module in the Crimson Summer Academy, a summer program at Harvard for academically talented high school students from financially disadvantaged backgrounds. This program will engage students with a wide range of topics including gene and genome organization, gene evolution and the process of writing a publishable scientific article.

Project Report

While evolutionary change is often marked by gradual transitions in morphology, the dramatic appearance of novel features, such as feathers, horns or flowers, has fascinated biologists for centuries. Of course, with molecular tools, researchers have been able to elucidate the genetic basis of the evolution of many novel features and one trend appears clear: novel features can be produced by modification of existing genetic modules rather than the de novo evolution of completely new genetic pathways. Investigating novelty is of wide interest given that the bioengineering of complex novel traits, such as C4 photosynthesis in crop plants or bioremediation capacity in weedy species, are the goals of many translational research programs. To be successful in such an effort, however, we need a model system in which novelty has arisen relatively recently and for which we possess the suitable molecular tools. These criteria have led us to the angiosperm model system Aquilegia (columbine), which possesses several distinct forms of recently evolved morphological novelties. Aquilegia is a dicot model being used to address diverse topics related to speciation and the evolution of floral development. The main attraction to Aquilegia is its unusual floral morphology (Fig. 1A). The outer sterile organs are composed of two concentric whorls of morphologically distinct petaloid organs: the first whorl petaloid sepals and the second whorl petals, which are characterized by large nectar spurs (Fig. 1A). Internal to the 4-7 whorls of stamens is one whorl of sterile organs termed staminodia, which are composed of a central filament flanked by lateral wings (Fig. 1B). It has been hypothesized that the staminodia play a role in protecting the young ovaries from herbivory damage. In contrast, the evolutionary significance of the nectar spur is readily apparent as a critical mediator of pollinator interactions. Previous studies have found that discrete shifts in spur length, as well as other pollinator-related features such as color and spur shape, are closely associated with speciation events in Aquilegia. In this project, we used a variety of tools to investigate these two distinct forms of floral novelty in Aquilegia: the petal nectar spur and a fifth type of floral organ, the staminodium. In the context of earlier funded work, we discovered that some of the genes that normally promote stamen identity in the flower (Fig. 1C) have experienced duplication in the lineage leading to Aquilegia such that there are three copies of a gene called AP3. AqAP3-1 experienced a process called neofunctionalization in which AqAP3-1 became uniquely associated with establishing the identity of the novel inner staminodia while AqAP3-2 remained as the primary stamen identity gene, thereby creating a distinct genetic code for the staminodia relative to the other floral organs (Fig. 1D). In addition, the third gene duplicate, AqAP3-3, has become specifically involved with promoting petal identity. In Aim 1 of award 1121005, we discovered that the differential expression of the three AP3 paralogs is at least partially controlled by distinct functions of a pair of genes called AqUFO1/2. In other dicot species, UFO orthologs play critical roles in activating the expression of AP3 homologs but in Aquilegia, it appears that the AqUFO1/2 loci only promote the expression of AqAP3-3. In parallel, we also studied the functions of genes that normally promote stamen and carpel identity, which have also duplicated in the lineage leading to Aquilegia to yield AqAG1 and AqAG2. We found that both of these genes contribute to staminodium identity but that while AqAG1 promotes stamen, staminodium and carpel identity, AqAG2 primarily acts in staminodium identity. This further elaborates the novel staminodium genetic program to include multiple gene duplicates. In Aim 2, we investigated additional candidates for control of staminodium identity and actually ruled out several players, although additional studies are underway to confirm this. Aim 3 of this project focused on the molecular basis of nectar spur development, which is the result of three-dimensional elaboration from a simple laminar primordium. Initiated by localized, oriented cell divisions surrounding the incipient nectary, this process creates a pouch that is then extended by anisotropic cell elongation. Using cell division markers to guide transcriptome analysis of microdissected spur sectors, we characterized candidate mechanisms underlying this three dimensional outgrowth. We see dynamic expression of factors controlling cell proliferation and hormone signaling, but no evidence of contribution from indeterminacy factors. Functional verification of a role for localized sculpting by Aquilegia TCP4 revealed an unexpected asymmetric component of spur development (Fig. 2). Transcriptome dynamics point to a novel recruitment event in which auxin-related factors that normally function at the organ margin were co-opted to this central structure. These findings indicate that spur development is an example of organ sculpting via localized cell division with novel contributions from hormone signaling.

National Science Foundation (NSF)
Division of Integrative Organismal Systems (IOS)
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Steven L. Klein
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Harvard University
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