Clay J. Carter, Ph.D. - US grants

DESCRIPTION (provided by applicant): All eukaryotes compartmentalize biomolecules within membrane bound organelles. The transport of these biomolecules (particularly proteins) to their final destination is dependant upon proper vesicle formation and transport. Many basic biological processes are thought to be controlled through vesicles, ranging from insulin signaling to memory development. Further, a number of diseases are manifested through the endomembrane system, including Alzheimer's disease and the pathogenesis of certain microorganisms. The basic mechanisms involved in vesicle trafficking appear to be conserved between yeast, plants and animals. The purpose of this proposal is to study the uncharacterized vesicle-dependent carboxy-terminal propeptide (CTPP) pathway to the protein storage vacuole (PSV) in plants. The proposed experiments will be performed in Arabidopsis thaliana and lead to the identification of the molecular machinery required for this trafficking process. Results from these experiments will further our understanding of vesicle mediated trafficking mechanisms in eukaryotes.

PI: Clay J. Carter (University of Minnesota-Twin Cities)

CoPIs: Robert W. Thornburg and Basil J. Nikolau (Iowa State University) and Marshall W. Hampton (University of Minnesota-Duluth)

Collaborators: Martin Heil (CINESTAV, Irapuato, Mexico) and Patrick von Aderkas (University of Victoria, Canada)

One-third of all crop species produce floral nectar and are dependent on animals for reproduction. Moreover, crops such as cotton, bean, pea, apple, cherry, peach, and blueberry, all produce extrafloral nectar to attract mutualistic predatory insects, which protect plants from herbivores. U.S. pollinator-dependent crops alone have an annual value of nearly $25 billion, and extrafloral nectar represents one of the few defense mechanisms for which stable effects on plant health and fitness have been demonstrated. This project will employ genomics technologies to study the molecular mechanisms involved in the synthesis and secretion of nectar components across species and nectary types. If successful, this project will provide significant new insight into these processes and the plant-animal interactions mediated by specific nectar components that will allow targeted studies to improve overall pollination efficiency, enhance biological control of pests that attack crops, and have the potential to greatly impact apiculture. With regard to outreach and training, the project will provide interdisciplinary research training for students and postdoctoral associates. Project graduate students at Iowa State University will also serve as GK12 Fellows in GK12 Symbi program (http://www.gk12.iastate.edu/). Supported by NSF, the Symbi program is a partnership between ISU and middle schools and high schools that serve primarily under-represented students in the Des Moines Public School District. GK12 Fellows will serve as "resident scientists" who will work with teachers to develop innovative, hands-on and engaging science activities for select middle school or high school science students. Finally, the project will also provide research experiences for high school students from Cornell High School (Cornell, Wisconsin).

The central hypothesis driving the project is that the genetic programs underlying nectar synthesis, secretion and function are at least partially conserved between diverse species and nectary types. To address this hypothesis, the project will apply comparative genomics approaches to the characterization of the (i) synthesis, (ii) secretion and (iii) biological function of nectar components across a diverse set of core eudicots, including Brassica sp., cotton, tobacco, squash, Lima bean, and acacia. The specific objectives and approaches include:

1. Comparative expression profiling of nectaries throughout the secretory process. Both the floral and extrafloral nectary transcriptomes and proteomes of the respective species will be examined at several time points throughout the secretory process;

2. Comparative metabolomics of nectars. Nectar metabolites will be comprehensively characterized through a non-biased GC-GC/MS-MS and LC/MS-MS approach;

3. Comparative and functional analysis of nectar proteins. Specific arrays of proteins have been reported in several nectars, which confer both protective and attractive properties. Thus, the proteomes of both floral and extrafloral nectars will be defined and functionally characterized across species.; and,

4. Informatics, database building & initiation of mechanistic studies. Mining of global expression data from actively secreting vs. inactive nectaries will allow the identification of genes and cellular processes central to the basal pathways for active nectar secretion across species. Metabolomic data will be linked to activities identified from expression data to assess their impacts on nectar production and composition.

All validated data will subsequently be integrated into an existing public online database (www.nectarygenomics.org), with subsets of data also being made available to the public at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) and the iPlant Collaborative (www.iplantcollaborative.org).

Nearly 90% of flowering plants produce floral nectar, the volume and composition of which have been correlated with the efficiency of animal-mediated pollination that impacts the production of 87 out of 115 (~76%) of the leading global food crops which represents an annual value of $29 billion in the U.S. alone and accounts for one-third of total food production. Although sugars are the predominant nectar solutes, ~10% of nectar dry weight is represented by many types of non-sugar solutes, including specialized metabolites, like pigments. Pigments play essential roles in biotic interactions throughout the Tree of Life, with natural pigments having a global market value of >$5B annually. This project will use a comparative genomics interdisciplinary approach to test the central hypothesis that colored nectars contain novel and known pigments, which evolved from being selectively favored through having spectral qualities attuned to pollinator vision. The activities outlined in this study are wholly novel and will provide new insights into the biology of an essential plant-animal mutualism on a genome-wide scale by (1) systematically profiling nectar pigments across species; (2) determining to what extent nectaries can actively control non-sugar nectar content, with an emphasis on nectar pigments; (3) understanding how evolutionary and natural history impact nectar quality with respect to pigment content; (4) identifying and characterizing enzymes involved in pigment synthesis; and, (5) systematically evaluating the true biological functions of nectar pigments. With respect to broader impacts, the project will help train the next generation of plant biologists by providing research training experiences for undergraduate, graduate, and middle school students. In addition, the project will take advantage of the broad public interest in the conservation of plant:pollinator interactions through citizen science projects.

Plant-pollinator interactions have at least partially driven the massive species radiation observed in the angiosperms, leading not only to extreme diversification in flower size and morphology, but also the accompanying chemistries behind attractants (color and scent) and rewards (nectar and pollen). Although rare, one floral trait that has evolved multiple times is colored nectar, which is suggested to serve as a visual cue of reward to prospective pollinators. Approximately 70 plant species are known to produce colored nectars, but the identities of these pigments, their associated syntheses, and true biological functions, have not yet been described. To address this gap in knowledge, the overarching goal of this project is to decipher the diversity, synthesis, and function of nectar pigments within a phylogenetic and evolutionary framework. The specific aims of this project include:

1) Systematic identification of nectar pigments in 20+ taxa of diverse lineages via LC MS/MS, including multiple sister taxa with different colored nectars.

2) Comparative transcriptomics and proteomics of nectaries and nectars. RNA-seq will be conducted on nectaries throughout floral maturation to enable comparative mining of pigment synthesis pathways, as well as nectar protein identification, which we predict to be involved in pigment formation.

3) Determine mechanisms of pigment formation. This aim will characterize the role of nectar proteins in pigment formation.

4) Initiation of functional analyses of nectar pigments. Nectar pigments may serve as visual cues to pollinators. This aim will evaluate if colored nectars are visible and conspicuous to suspected pollinators.

All methods and datasets will be made publicly available through the project website (www.nectarygenomics.org) and publications. Long-term access will be provided through established public data repositories.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.