Generating and Maintaining Species Boundaries
Summary: Daphne Preuss is investigating the cellular machinery that evolves rapidly as species diverge, including the DNA and proteins that mediate chromosome segregation, the genes that control reproduction, and the signals that trigger allergy.
To maintain species identity, most organisms exercise elaborate controls to ensure that their offspring inherit the correct genetic material. DNA strands are partitioned into daughter cells during every cell division with astonishing precision, providing gametes with an appropriate chromosome complement. Furthermore, both sperm and egg cells often exhibit remarkable selectivity, fusing only with appropriate gametes of their own species. When new species emerge, the machinery that regulates inheritance diverges rapidly, creating barriers that prevent cross-species hybridization. At the same time, the newly derived species undergo efficient chromosome segregation and reproduction. Recent discoveries have highlighted how rapidly this evolution can take place; even so, the mechanisms that balance the selection for divergence with the requirement for high-fidelity inheritance are not understood.
To investigate the forces driving speciation, we have selected a series of nondomesticated plants from the mustard (Brassicaceae) family, of which the model organism, Arabidopsis thaliana, is a member. In the evolutionary time frame of primate diversification (~40-50 million years, MY), this family has expanded to 3,350 species, providing a substantial resource for exploring evolutionary change. To date, we have examined rapidly evolving genes and chromosomal regions from species separated from A. thaliana by 5 MY (A. arenosa), 10-14 MY (Capsella rubella and Olimarabidopsis pumila), and 16-21 MY (Sisymbrium irio). Our investigations of plant reproduction have defined signaling events, novel assays, and genes that evolve rapidly. We are extending our studies of pollen surface proteins to more distantly related species that play a role in allergy, such as birch, cottonwood, and ragweed.
Pollen Diversity and Mating Interactions
Plants invest enormous resources to ensure that their sperm and egg cells interact with the most suitable partners. Adhesion between pollen and the stigma occurs within seconds of pollination, is extremely strong, and highly species specific. Subsequently, the pollen undergoes striking cellular changes, mobilizing its extracellular matrix to move onto the stigma surface, and forming a polar process (the pollen tube) that delivers the sperm to the ovary. Our research has defined key reproductive signals, characterizing pollen surface components, pollen wall structure, and factors contributing to pollen tube growth and guidance. Each of these processes undergoes substantial divergence between even closely related species, suggesting that mate specification results from the sum of multiple barriers. Ultimately, our investigations will improve our ability to predict, and even manipulate, gene flow between species, facilitating the creation of new hybrids and the containment of genetically modified crop varieties.
Pollen recognition at the stigma surface. Brassicaceae pollen and stigma cells interact in a nonaqueous environment—the stigma is covered with a waxy cuticle, the hydrophobic pollen coat proteins are embedded in a lipid matrix, and the pollen wall is composed of an inert hydrophobic polymer. Nonetheless, interactions are sufficiently precise to promote the germination of only a subset of pollen grains. To understand the specificity of these interactions, we have examined the composition of the pollen and stigma surfaces, defining lipids, proteins, and cell wall constituents. Specifically, we have found mutations that alter pollen adhesion, characterized the pollen coat proteome and its divergence across Brassicaceae species, and defined genes differentially expressed in stigmas. We now have sufficient resources to begin replacing A. thaliana genes with those of its relatives, with the ultimate goal of converting A. thaliana reproductive identity to that of another species.
Pollen-stigma adhesion is rapid, extremely strong, highly selective, and mediated by exine, the outer pollen wall. Exine patterns are so variable among species that they serve as taxonomic and forensic tools. These patterns arise from the self-assembly of sporopollenin, a chemically inert polymer of unknown structure. Thus, although exine plays one of the earliest, and perhaps most important roles in mating specificity, it is poorly understood. We are using a genetic strategy to investigate exine development—although male-sterile mutations that grossly alter exine structure were identified previously, our screen for nonadhesive pollen uncovered defects that are more subtle. Cloning these genes has implicated alkaloid, fatty acid, and phenylpropanoid biosynthesis pathways in exine patterning. Using reverse genetic approaches, we are characterizing the network of genes involved in exine biosynthesis, assembly, patterning, and adhesion. Surveys of public sequence databases will aid in assessing whether rapid evolution of the exine assembly genes implies positive selection for divergence. (This effort is supported in part through the National Science Foundation 2010 Program.)
Pollen tube guidance. Pollen tube navigation relies on attractive, repulsive, and adhesive interactions analogous to the cues that guide axons to neural synapses. These barriers provide redundancy that ensures only compatible pollen succeeds, and we are exploring the signals exchanged between pollen tubes, ovule tissues, and haploid female gametophytes. We recently developed an in vitro system to discern the temporal, spatial, and genetic regulation of guidance signals and pollen tube responses. (This work was supported in part by the Department of Energy.) Interspecies pollinations between A. thaliana and its relatives previously showed that pollen tube guidance signals diverge rapidly, showing random and arrested pollen tube growth with ~25-MY separation. Using the in vitro assay, we found that A. thaliana pollen tubes inefficiently target ovules from A. arenosa (separated by 5 MY), rarely target O. pumila ovules (10 MY), and fail to target C. rubella or S. irio ovules (10 and 20 MY, respectively), yet pollen tube guidance is insensitive to the origin of tissue from the stigma or style. Unlike the proposed calcium signals that emanate from female gametophytes, our results point to a diffusible, heat-labile, ovule-derived signal that is sufficiently complex for rapid evolution—criteria that are most consistent with a protein-based signal. Our in vitro system also uncovered strong evidence for a repulsive signal that prevents ovules from being fertilized multiple times; although such a signal was postulated from in vivo observations, pollen tubes undergoing repulsion had not been previously observed.
Pollen Surface Diversity and Allergy
Our characterization of the pollen surface has provided unexpected insight into its role in triggering allergy and asthma. Surprisingly, previous efforts to identify pollen allergens inadvertently focused on the pollen cytoplasm, overlooking the hydrophobic surface. With asthma and allergy on the rise, improved diagnostics and therapeutics are badly needed. Immunotherapy remains one of the most promising avenues for long-term allergy relief, yet clinical protocols for allergen extraction result in immunotherapeutics that lack pollen coat material.
We are examining candidate pollen surface allergens and exploring the potential for pollen lipids to trigger inflammatory or innate immune responses. By purifying pollen coat lipids and proteins from 22 allergenic species, we have identified a diverse set of compounds. Hydrophobic small molecules from the pollen surface can stimulate immune cell responses, including migration and cytokine production. In addition, we have probed cytoplasmic and coat pollen fractions with human sera, demonstrating that for some pollens, the human population has higher levels of IgE directed against the pollen coating. We have used this assay to clone novel allergens. (This research is supported in part by the Sandler Program for Asthma Research.)