Plant microRNAs have been shown to have important roles in regulating diverse processes ranging from reproductive development to stress response. In the first two chapters, I focus on miRNA diversity in Aquilegia studying both anciently evolved broadly conserved and rapidly evolving species specific miRNAs. In chapter one, I utilize Aquilegia's critical phylogenetic position between the well developed models Arabidopsis thaliana and Oryza sativa to study the evolution of ancient miRNAs across the angiosperms. In chapter two, I utilize smallRNA high-throughput sequencing to annotate Aquilegia specific miRNAs and, in the process, uncover the novel regulation of a floral homeotic gene by an Aquilegia-specific miRNA. In chapter three, I look at the tissue specific development of miRNA regulation in the bioenergetically relevant model organism Populus trichocarpa. High-throughput smallRNA sequencing from four diverse tissue sets including leaves, xylem, mechanically treated xylem, and pooled vegetative and reproductive tissues were analyzed, revealing a total of 155 previously unannotated miRNAs, most of which are P. trichocarpa specific. Expanding on my work with the petal identity pathway, I turned a broader analysis of Aquilegia petal spurs. Petal spurs are the distinguishing characteristic of Aquilegia and are argued to be a key innovation in the adaptive radiation of the genus. In the fourth chapter, I explore the cellular basis of extreme spur length diversity in the genus and find that a single parameter, cell shape, can explain this morphological range. Next, I seek to describe the cellular patterns that give rise to a spur primoridia from an initially flat laminar petal and find that spur initiation is characterized by concentrated, prolonged, and oriented cell divisions. Inspired by this quantitative analysis of growth, chapter five looks at the mechanisms of shape change in cucumber tendrils. I find that anisotropic contraction of a multi-layered gelatinous fiber ribbon explains coiling in cucumbers. Surprisingly, we discover that tendrils display twistless-overwinding when pulled and exhibit an unforeseen force-extension response as a result. These results provide the design basis for twistless springs with tunable mechanical responses and serve as a clear example of how the biological systems can inspire applied mechanical designs.

Epichloë species and their anamorphs, Neotyphodium species, are fungal endophytes that inhabit cool-season grasses and often produce bioprotective alkaloids. These alkaloids include lolines, which are insecticidal and insect feeding deterrents. Lolines are exo-1-aminopyrrolizidines with an oxygen bridge between carbons 2 and 7, and are usually methylated and formylated or acetylated on the 1-amine. In previously published studies lolines were shown to be derived from the amino acids L-proline and L-homoserine. In addition the gene cluster involved in loline-alkaloid biosynthesis has also been characterized. In this dissertation a survey of plant-endophyte symbioses revealed a phenotype with only N-acetylnorloline. This phenotype provided insights into loline alkaloid production. This dissertation focuses on determining the steps to loline biosynthesis after the amino acid precursors. The study involves feeding isotopically labeled potential precursors to loline-alkaloid-producing cultures of Neotyphodium uncinatum, as well as RNA interference (RNAi) of N. uncinatum genes for steps in the pathway. Synthesized deuterated compounds were fed to loline-alkaloid-producing cultures of N. uncinatum to test their possible roles as precursors or intermediates in the loline-alkaloid pathway. N-Formylloline was extracted from the cultures and assayed by GCMS for incorporation of the deuterium label. The results indicated that N-(3-amino, 3-carboxy)propylproline and exo-1-aminopyrrolizidine are intermediates in the loline-alkaloid biosynthetic pathway. Plasmids were also designed for expression of double-stranded RNA homologous to loline-alkaloid biosynthesis genes, and introduced by transformation into N. uncinatum. This RNAi strategy resulted in fungal transformants altered in loline-alkaloid profiles. The RNAi results indicated that N-acetyl-1-aminopyrrolizidine is the intermediate before oxygen bridge formation. Based on the results of this study and the likely roles of the loline-alkaloid biosynthesis genes inferred from signature sequences of their predicted protein products, I propose a pathway of bond formation steps in loline-alkaloid biosynthesis.

Sterols have been identified as major components of membrane lipids that are part of specialized membrane domains necessary for organizing events such as polar protein targeting and signal transduction in plants, fungi and animals. However a common modification of sterols is the addition of sugar moieties via glycosylation abundantly found in plants. An exact physiological role for such diversification of sterols in plants is still unknown. Using reverse genetics and transcriptomics we show that UDP-glucose: sterol glucosyltransferase encoded by UGT80B1 is necessary for correct epidermal patterning in Arabidopsis root. Patterning of hair cells (trichoblasts) and non-hair cells (atrichoblasts) in the epidermis of the Arabidopsis root involves signaling through SCRAMBLED (SCM), a plasma membrane localized LRR-RL kinase. Feedback regulation via the transcriptional regulatory complex containing R2R3-MYB transcription factor WEREWOLF (WER) represses SCM and activates the homeodomain-leucine-zipper protein GLABRA2 (GL2) in atrichoblasts. Evidence suggests symplastic connections between cells, known as plasmodesmata, establish passage ways for single-repeat R3-MYB transcription factors to activate SCM expression in trichoblasts. Mutations in UGT80B1 cause atypical localization patterns of GL2, WER, and SCM in the root epidermis. The ugt80B1 formed fewer trichoblasts in comparison to wild-type. A translational fusion of UGT80B1 to GFP localizes to the ER, plasma membrane and to sites that appear to be plasmodesmata-associated desmotubules. Ultrastructural analysis revealed abnormalities in plasmodesmata formation and morphology in ugt80B1 mutants. Steryl glucoside profiling indicated deficiencies in specific glycosylated sterol compounds in roots. This study identifies UGT80B1 as a novel membrane component that is critical for plasmodesmata morphogenesis and cell-fate determination in the root epidermis. A model is proposed in which UGT80B1 activity provides spatially discreet sterol and steryl glucoside architecture within the plasma membrane to anchor the SCM receptor and within plasmosdesmata to facilitate intercellular movement of R3-MYB regulatory proteins underlying proper differentiation of trichoblasts versus atrichoblasts. Moreover, evidence from reverse genetics, proteomics and live cell imaging point to a actin dependent localization of UGT80B1 at the vesicle rich zone of root hair tip. This localization actively supports root hair elongation via tip growth, possibly by membrane modifications required for vesicle trafficking.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Plant Biology." Includes bibliographical references.

abstract: In the United States, Escherichia coli O157:H7 (E. coli O157:H7) is the most frequent cause of hemolytic uremic syndrome (HUS) and it is also the primary cause of acute renal failure in children. The most common route of the infection is ingestion of contaminated meat or dairy product originating from cattle or vegetables contaminated with bovine manure. Since cattle are the main reservoir for human infection with E. coli O157:H7, the reduction of intestinal colonization by these bacteria in cattle is the best approach to prevent human infections. Intimin is an outer membrane protein of E. coli O157:H7 that plays an important role in adhesion of the bacteria to the host cell. Hence, I proposed to express intimin protein in tomato plants to use it as a vaccine candidate to reduce or prevent intestinal colonization of cattle with E. coli O157:H7. I expressed His-tagged intimin protein in tomato plants and tested the purified plant-derived intimin as a vaccine candidate in animal trials. I demonstrated that mice immunized intranasally with purified tomato-derived intimin produced intimin-specific serum IgG1and IgG2a, as well as mucosal IgA. I further demonstrated that mice immunized with intimin significantly reduced time of the E. coli O157:H7 shedding in their feces after the challenge with these bacteria, as compared to unimmunized mice. Shiga toxin is the major virulence factor that contributes to HUS. Since Shiga toxin B subunit has an important role in the attachment of the toxin to its receptor, I fused intimin to Shiga toxin B subunit to create multivalent subunit vaccine and tested the effects upon immunization of mice with the B subunit when combined with intimin. His-tagged intimin, Shiga toxin B subunit, and Shiga toxin-intimin fusion proteins were expressed in E. coli and purified. I demonstrated that this multivalent fusion protein vaccine candidate elicited intimin- and Shiga toxin B-specific IgG1, IgG2a, and IgA antibodies in mice. I also showed a reduction in the duration of the bacterial shedding after the challenge compared to the control sham-immunized groups. / Dissertation/Thesis / Ph.D. Plant Biology 2010

abstract: ABSTRACT The Phoenix Four Rivers Flora is an inventory of all the vascular plants growing along the Salt, Gila, New and Agua Fria Rivers, and their tributaries in the Phoenix Metropolitan Area during the years of the study (2009-2011). This floristic inventory documents the plant species and habitats that exist currently in the project area, which has changed dramatically from previous times. The data gathered by the flora project thus not only documents how the current flora has been altered by urbanization, but also will provide a baseline for future ecological studies. The Phoenix Metropolitan Area is a large urbanized region in the Sonoran Desert of Central Arizona, and its rivers are important for the region for many uses including flood control, waste water management, recreation, and gravel mining. The flora of the rivers and tributaries within the project area is extremely diverse; the heterogeneity of the systems being caused by urbanization, stream modification for flood control, gravel mining, and escaped exotic species. Hydrological changes include increased runoff in some areas because of impermeable surfaces (e.g. paved streets) and decreased runoff in other areas due to flood retention basins. The landscaping trade has introduced exotic plant species that have escaped into urban washes and riparian areas. Many of these have established with native species to form novel plant associations. / Dissertation/Thesis / M.S. Plant Biology 2011

abstract: The cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged from 1-33 and 30-75 hours, respectively. Interestingly, chlorophyll had longer lifetimes than the chlorophyll-binding proteins in these photosystems. Therefore, photosynthetic proteins turn over and are replaced independently from each other, and chlorophyll is recycled from the damaged chlorophyll-binding proteins. In Synechocystis, there are five small Cab-like proteins (SCPs: ScpA-E) that share chlorophyll a/b-binding motifs with LHC proteins in plants. SCPs appear to transiently bind chlorophyll and to regulate chlorophyll biosynthesis. In this study, the association of ScpB, ScpC, and ScpD with damaged and repaired PSII was demonstrated. Moreover, in a mutant lacking SCPs, most PSII protein lifetimes were unaffected but the lifetime of chlorophyll was decreased, and one of the nascent PSII complexes was missing. SCPs appear to bind PSII chlorophyll while PSII is repaired, and SCPs stabilize nascent PSII complexes. Furthermore, aminolevulinic acid biosynthesis, an early step of chlorophyll biosynthesis, was impaired in the absence of SCPs, so that the amount of chlorophyll in the cells was reduced. Finally, a deletion mutation was introduced into the sll1906 gene, encoding a member of the putative bacteriochlorophyll delivery (BCD) protein family. The Sll1906 sequence contains possible chlorophyll-binding sites, and its homolog in purple bacteria functions in proper assembly of light-harvesting complexes. However, the sll1906 deletion did not affect chlorophyll degradation/biosynthesis and photosystem assembly. Other (parallel) pathways may exist that may fully compensate for the lack of Sll1906. This study has highlighted the dynamics of photosynthetic complexes in their biogenesis and turnover and the coordination between synthesis of chlorophyll and photosynthetic proteins. / Dissertation/Thesis / Ph.D. Plant Biology 2011

abstract: Acceptance of the plant group Martyniaceae as a distinct family has long been questioned. Previously placed in the family Pedaliaceae, the Martyniaceae have been allied to numerous other families within the order Lamiales. The objectives of this study include the investigation of the placement of the Martyniaceae within the order Lamiales using molecular data (chloroplast DNA sequences), the further examination of the internal relationships of the Martyniaceae using an expanded nuclear and chloroplast sequences data set, and the construction of a taxonomic treatment of the family that includes all published names and taxa in the Martyniaceae. An analysis of the Lamiales using two chloroplast gene regions (ndhF and rps16) reveals that the Martyniaceae should be segregated from the family Pedaliaceae, but is not able to support the placement of any of its putatively-related families as sister to the Martyniaceae. Sequences from 151 taxa of the Lamiales are included in the analysis, including six representatives from the Martyniaceae. An analysis of the Martyniaceae using three chloroplast gene regions (psbA-trnH spacer, trnQ-5'rps16 intergenic spacer, and trnS-trnG-trnG spacer and intron) and the Internal Transcribed Spacer resolves two major clades within the Martyniaceae corresponding to the North American taxa (Martynia and Proboscidea) and the South American taxa (Craniolaria, Holoregmia, and Ibicella). Sequences from all five genera and 15 taxa were included in the analysis. Results from the molecular phylogenetic analyses are incorporated into a revised taxonomic treatment of the family. Five genera and thirteen species are recognized for the family Martyniaceae. / Dissertation/Thesis / Ph.D. Plant Biology 2011

abstract: The Zingiberales, including the gingers (Zingiber), bananas (Musa) and ornamental flowers (Strelitzia, Canna, and Heliconia) are a diverse group of monocots that occupy the tropics and subtropics worldwide. The monophyly of the order is well supported, although relationships between families are not well resolved. A rapid divergence of the Zingiberales has been proposed to explain the poor resolution of paraphyletic families in the order, and direct fossil evidence shows members of both of these lineages of Zingiberaceae and Musaceae were present by the Late Cretaceous. Comparisons of the fossils with extant relatives and their systematic placement have been limited because variation within modern taxa is not completely known. The current study focuses on describing zingiberalean fossil material from North Dakota that includes seeds, leaves, buds, adventitious roots and rhizomes. A survey of extant zingiberalean seeds was conducted, including descriptions of those for which data were previously unknown, in order to resolve the taxonomic placement of the fossil material. Upon careful examination, anatomical characters of the seed coat in fossil and extant seeds provide the basis for a more accurate taxonomic placement of the fossils and a better understanding of character evolution within the order. / Dissertation/Thesis / Ph.D. Plant Biology 2012