It is critically important that eukaryotic cells possess the capacity to undergo intracellular change in response to both internal and external cues. Arabidopsis thaliana F-box (FBX) and 14-3-3 proteins are two distinct protein families that regulate intracellular change and have important roles in regulating responses to environmental stress. FBX proteins are variable substrate adaptors within the Ubiquitin 26S Proteasome System (UPS), a system the selects proteins to be degraded by the 26S proteasome using selective ubiquitylation. 14-3-3 proteins recognize and bind unique phosphorylated residues in client proteins to regulate subcellular localization, inhibit protein interactions, and act as a scaffold to facilitate protein interactions. Thus, these two protein families independently have the ability to drastically alter cell physiology. 14-3-3 proteins have been shown to interact with components of the Ubiquitin 26S Proteasome System in only a few instances, including their interaction with the F-box protein F-BOX STRESS INDUCED 1 (FBS1). However, the biological consequences of this FBS1:14-3-3 interaction are completely unknown. FBS1 belongs to a four-member FBX protein family, along with FBS2-4, and we hypothesize that 14-3-3 proteins more broadly interact to at least regulate this family of four proteins. A Yeast 2-Hybrid assay has been employed to test for interactions between FBS1-4 and five of thirteen different 14-3-3 proteins. Full length and truncated constructs are currently being tested to probe the extent of interactions between these two important protein families. Interaction between multiple members of these two important protein families could point to a more broadly used scheme in regulating intracellular events in plant cells.

TOPLESS (TPL) is a transcriptional co-repressor protein that plays a central role in the regulation of plant growth and development. In the model plant Arabidopsis thaliana, TPL acts in essential hormone response pathways, including that of auxin, a small signaling molecule. TPL acts in the auxin response pathway by binding another co-repressor from the Aux/IAA family (henceforth referred to as IAAs). In the presence of auxin, IAAs are degraded, relieving TPL repression and allowing for transcription of auxin responsive genes. Despite its central role, the molecular mechanism by which TPL confers repression is not well understood. Recent structural analyses indicate that TPL has interfaces for both homodimerization and homotetramerization. Synthetic assays have shown that TPL truncations in which the tetramerization interface has been deleted have a significant decrease in repressive function. To determine the repressive mechanisms of TPL, we created a structure-function map. We have created full-length TPL variants with point mutations at the dimerization and tetramerization interfaces, as well as serial domain deletions. We tested the repressive function of these variants in a yeast synthetic assay in which TPL represses expression of a fluorescent protein. Repressive function was then quantified from fluorescent reporter output with stronger repression corresponding to decreased fluorescence. We then validated the results of our synthetic assays in planta by expressing TPL-IAA14 variants that negatively regulate lateral root development. The repressive strength of each TPL variant will be quantified by measuring the number of lateral roots, with fewer lateral roots corresponding to increased TPL repressive strength. TPL homologs exist in many species. Defects in TPL homologues have been implicated in the causes of many cancers and developmental diseases, therefore a better understanding of the functional mechanisms of TPL will have broad implications across organisms, including humans.

Auxin is a hormone that is crucial for plant growth and development. Auxin promotes the activity of auxin responsive transcription factors (ARFs) that induce transcription of target genes. Though, we know a lot about auxin response in the model plant Arabidopsis, very little is known about ARF behavior in maize. It has been previously shown that auxin and ARFs mediate many diverse developmental responses in Arabidopsis and maize. We hypothesize this diversity of response may be caused by different ARFs binding to different promoters, allowing different genes to be activated. The goal of the project is to examine the activity of maize ARFs on different promoter structures. We tested if maize ARFs show similar rules of ARF-promoter interactions as Arabidopsis ARFs. By comparing Arabidopsis and maize ARF activity, we are be able to understand how the two species have evolved over time, and if ARF function and auxin response are conserved in the two species. Working on maize is also important because it such an essential food crop for people and animals and can help us resolve issues such as food shortage. To investigate how ARFs regulate the activation of genes, we used a synthetic yeast system to quantify transcriptional activation. We engineered yeast to express both a maize ARF and a synthetic auxin-responsive promoter regulating transcription of a fluorescent reporter. We quantified fluorescence, a readout of ARF activation of the promoter, by flow cytometry. We systematically tested how activation is impacted by the sequence, orientation, and number of cis-elements within a promoter sequence, and how these effects differ among different ARFs.

Flowering plants exhibit striking diversity in their floral symmetry due to independent genetic changes throughout their evolutionary history. Diversity is also observed within the flowers of genus Rhododendron, which displays a variety of radially and bilaterally symmetric flowers across its approximately 1000 species. Floral symmetry is governed by an interaction between one TCP transcription factor, CYCLOIDEA (CYC), and two MYB-class genes, RADIALIS (RAD) and DIVARICATA (DIV). This interaction initiates when CYC is expressed in the floral meristem, leading to the activation of RAD in the dorsal domain. RAD then antagonizes DIV activity in the dorsal regions, leading to radial symmetry. In other flowering plants, duplications in CYC have been associated with changes in floral symmetry, but the role of RAD and DIV remain unstudied. A number of transitions to and reversals from radial symmetry have occurred throughout Rhododendron from a bilaterally symmetric ancestor. Focusing on RAD and DIV, the goal of this study is to better understand the evolutionary history of these floral symmetry genes in rhododendrons, and their correlation to floral symmetry changes. I sampled eight different species throughout Rhododendron in addition to various outgroups and reconstructed phylogenies of these genes. I obtained sequences from genomic and transcriptomic databases, and used these sequences to design primers for amplifying and sequencing these genes from laboratory samples from wild and cultivated specimens. I found two distinct copies resulting from one duplication, in both RAD and DIV. In future studies, we will expand our sampling of species to investigate the phylogenetic placement of the origin of these duplications in tandem with changes in flower symmetry, to determine whether duplications in RAD or DIV are associated with symmetry changes as has been shown for CYC.

Palms are a keystone species in tropical rainforests and are thought of as a model species due to their abundance and taxonomic diversity. Because over 90% of palm species diversity is concentrated in the tropical and subtropical biomes today, palm fossils are traditionally interpreted as indicators of warm and wet climate. However, recent work suggests that, in the past, palms may have played an important role also in other biomes. Palms produce great amounts of phytoliths (hardened silica bodies precipitated in and around plant cells), which, after the plant dies, are incorporated into the soil or sediment and preserved as fossils. Importantly, phytoliths are found in many environments where leaf impressions and other macrofossil are typically absent. Palm phytoliths are therefore a valuable potential source of information to reconstruct phylogenetic relationships and interpret the ecology of fossil palms and, by inference, paleoenvironments.Unfortunately, very little work has been done to comprehensively assess how much information about palm taxonomy and ecology can be inferred by palm phytolith morphology. To remedy this, we are conducting a morphometric study of palm phytoliths across the palm phylogenetic tree. We collected leaf samples from 80 palm taxa from herbaria and botanical gardens, then extracted phytoliths using strong oxidants and acids to decompose organic materials. Through measurement of their overall shape and size, as well as the density, size, and shape of phytolith ornamentation we hope to improve overall taxonomic resolution of the palm fossil record. In addition, for each of the taxa we recorded several habitat, climate, and ecology variables to test for biogeographical and ecological signal in palm phytolith morphology.