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Most gymnosperms produce a pollination drop that captures and transports pollen into the ovule. Pollination drops have other functions. These include influencing pollen germination and pollen tube growth, defending the ovule from pathogens and providing a food reward in insect-pollinated gymnosperms. Mineral and organic molecules, including proteins, are responsible for these additional functions. To date, pollination drops from a handful of conifers and one non-conifer gymnosperm, Welwitschia mirabilis, have been subjected to proteomic analysis. In the present study, tandem mass spectrometry was used to detect proteins in all gymnosperm lineages: cycads (Ceratozamia hildae, Cycas rumphii, Zamia furfuracea); Gnetales (Ephedra compacta, E. distachya, E. foeminea, E. likiangensis, E. minuta, E. monosperma, E. trifurca; Gnetum gnemon; Welwitschia mirabilis); Ginkgo biloba; conifers (Taxus x media). PEAKS 6 DB (Bioinformatics Solutions, Waterloo, ON, Canada) was used to make protein identifications. Proteins were detected in all gymnosperm species analyzed. The numbers of proteins identified varied between samples as follows: one protein in Welwitschia female; nine proteins in Cycas rumphii; 13 proteins on average in Ephedra spp.; 17 proteins in Gnetum gnemon; 38 proteins on average in Zamia furfuracea; 57 proteins in Ginkgo biloba; 61 proteins in Ceratozamia hildae; 63 in Taxus x media; 138 proteins in Welwitschia male. The types of proteins identified varied widely. Proteins involved in carbohydrate modification, e.g. galactosidase, chitinase, glycosyl hydrolase, glucosidase, were present in most gymnosperms. Similarly, defence proteins, e.g. reduction-oxidation proteins, lipid-transfer proteins and thaumatin-like proteins, were identified in many gymnosperms. Gymnosperms that develop a deep pollen chamber as the nucellus degrades, e.g., cycads, Ginkgo, Ephedra, generally contained higher proportions of proteins localized to intracellular spaces. These proteins represent the pollination drop degradome. Gymnosperms that either lack a pollen chamber, e.g. Taxus, or have a shallow pollen chamber, e.g. Gnetum, had greater proportions of extracellular proteins. These proteins represent the pollination drop secretome. Our proteomic analyses support the hypothesis that the pollination drops of all extant gymnosperms constitute complex reproductive secretions. / Graduate

In the first part of this research, we constructed a Genome scale Metabolic Model (GEM) of Taxus cuspidata, a medicinal plant used to produce paclitaxel (Taxol®). The construction of the T. cuspidata GEM was predicated on recent acquisition of a transcriptome of T. cuspidata metabolism under methyl jasmonate (MJ) elicited conditions (when paclitaxel is produced) and unelicited conditions (when paclitaxel is not produced). Construction of the draft model, in which transcriptomic data from elicited and unelicited conditions were included, utilized tools including the ModelSEED developed by Argonne National Laboratory. Although a model was successfully created and gapfilled by ModelSEED using their software, we were not able to reproduce their results using COBRA, a widely accepted FBA software package. Further work needs to be done to figure out how to run ModelSEED models on commonly available software. In the second part of this research, we modeled the MJ elicited/defense response phenotype in Arabidopsis thaliana. Previously published models of A. thaliana were tested for suitability in modeling the MJ elicited phenotype using publicly available computation tools. MJ elicited and unelicited datasets were compared to ascertain differences in metabolism between these two phenotypes. The MJ elicited and unelicited datasets were significantly different in many respects, including the expression levels of many genes associated with secondary metabolism. However, it was found that the expression of genes related to growth and central metabolism were not generally significantly different for the MJ+ and MJ- datasets, the pathways associated with secondary metabolism were incomplete and could not be modeled, and FBA methods did not show the difference in growth that was expected. These results suggest that behavior associated with the MJ+ phenotype such as slow growth and secondary metabolite production may be controlled by factors not easily modeled with transcriptome data alone. Additional research was performed in the area of cryosectioning and immunostaining of fixed Taxus aggregates. Protocols developed for this work can be found in Appendix B.

Indigenous landscape management has transformed ecosystems for millennia, with long-lasting impacts on the productivity and abundance of plant species. While western science based ecological research is beginning to investigate these impacts, less abundant species of cultural importance remain understudied. Pacific yew (Taxus brevifolia Nutt.), an uncommon understory conifer found in old-growth forests of the Northwest Coast of North America, has not received sustained ecological interest despite its importance to Indigenous Peoples throughout its range. In the first chapter, I synthesize the current ethnobotanical and ecological literature discussing Pacific yew to identify knowledge gaps and dominant paradigms that have shaped the study of the species. I find that many mechanisms behind Pacific yew’s habitat selection and ecosystem functions are unknown to western science and that the impacts of Indigenous landscape management are largely unacknowledged within the western scientific literature. In the following chapter, in partnership with the Heiltsuk First Nation, I examine the growth and abundance of Pacific yew on sites that were inhabited intensively by First Nations on the Central Coast of British Columbia for over 10,000 years. I find that habitation histories are not a strong driver of patterns of tree size and that Pacific yew abundance is largely driven by site aspect. These findings shed light on the habitat preferences of Pacific yew, which have rarely been studied in this region. They also illustrate variation in the response of culturally important species to landscape modification and highlight the need for nuanced understanding of the diversity of plant management strategies employed by Indigenous Peoples. This work is part of a broader attempt to incorporate cultural histories and questions into ecological study and to recognize the continuing ecological influences of Indigenous Peoples, who have stewarded their homelands for millennia. / Graduate

Abstract I hela Europa har idegranen problem med återväxten. Olika återväxthämmande faktorer påverkar föryngringen i olika stadier, och det är aldrig bara en anledning till att föryngringen inte lyckas. Idegranen är skuggtålig men behöver ljus. Den gynnas av lövfällande bestånd då den drar nytta av vårens öppna krontak. I bristen på ljus stannar de yngre plantorna av i utvecklingen, dessa dör eller äts upp och föryngringen uteblir. Nästan alla naturligt förekommande äldre plantor (> 2 år) betas. Bete av idegran är idag ett stort problem för föryngringen då rådjurens matsmältning klarar av idegran. I Stockholms norra skärgård är det framför allt betet som hämmar plantornas utveckling. Idegranen har problem med sin återväxt och detta sägs vara ett karaktärsdrag för arten.

Almost all conifer ovules produce a liquid secretion as part of reproduction. This secretion, termed an ovular secretion, is produced during ovule receptivity and is involved in pollen capture and transport. Historically, examinations of the ovular secretion have focused on how they are part of pollination mechanisms. As a result, the chemical composition of the ovular secretion has not been examined systematically. Investigations into the constituents of the ovular secretion were limited to analyses for simple water soluble compounds such as sugars, minerals, amino acids and organic acids. More recently, the protein component of the secretion has been investigated using mass spectrometry-based proteomics. Proteins involved in processes such as carbohydrate modification, proteolysis, and defence have been identified in conifer ovular secretions. This biochemical complexity suggests a broader view of the function of the ovular secretion is warranted. However, protein identifications only provide putative information on function. Functional characterization of these proteins is needed in order to fully understand how they contribute to ovular secretion function. The research outlined in this dissertation describes the first functional characterizations of proteins found in conifer ovular secretions. Three proteins - invertase, chitinase, and thaumatin-like protein - were characterized in the ovular secretions of Douglas-fir (Pseudotsuga menziesii) and hybrid yew (Taxus × media). The Douglas-fir ovular secretion is capable of converting sucrose to glucose and fructose, confirming that invertases present in the secretion are functional. The invertase activity was maximal at pH 4.0. Activity was 77% of maximal at pH 4.5, the physiological pH. This indicates that post-secretory hydrolysis of sucrose occurs in situ in the Douglas-fir ovular secretion. Invertases in the ovular secretion are likely involved in controlling the movement of carbohydrates to developing pollen and could facilitate pollen selection. Chitinases present in the Douglas-fir ovular secretion are functional at physiological conditions. All three modes of chitinolytic activity, i.e. endochitinase, chitobiosidase and β-N-acetylglucosaminidase, were detected at physiological pH. β-N-acetylglucosaminidase activity was 80 % of maximal at physiological pH. Chitinases are pathogenesis-related proteins capable of hydrolysing chitin in fungal cell walls. These results suggest the ovular secretion is capable of defending the ovule against infection by phytopathogens. Thaumatin-like protein was immunolocalized to the cell wall and amyloplasts in Douglas-fir and yew nucellar tissue in a pattern consistent with a defensive role. It was also localized to the cell wall of fungal spores and germinating hyphae that were present in the micropyle of a yew ovule. These results provide additional evidence for an antifungal role for the ovular secretion. Functioning enzymes involved in pollen-ovule interactions and ovule defence are present in the conifer ovular secretion. The ovular secretion has functions beyond pollen capture. A revised functional model for the conifer ovular secretion is proposed. / Graduate / 2021-08-17

Secondary metabolism in plants produces numerous compounds with wide-ranging activities, including the antineoplastic compound taxol and related taxanes. The biotechnological production of taxol has so far been based on empirical studies. The aim of the present work has been to study how the factors that enhance taxane production affect the metabolic profiles and gene expression in productive cells. As a consequence of this work, new potential candidates have been obtained for unknown taxane biosynthetic genes, some bottle-neck steps of taxane biosynthesis (in vitro and in silico) have been identified and a master regulator, not only for taxane biosynthesis, but also for other secondary metabolism routes, has been characterized. Coronatine, a powerful and less harmful elicitor than methyl jasmonate, has been successfully assayed and found to increase taxane production. In the different studies of this work, the expression level of genes that participate in taxol biosynthesis has been determined, clarifying their involvement in the production of this anti-cancer agent. / El metabolismo secundario de las plantas produce numerosos compuestos con un amplio rango de actividades, entre los que se encuentra el compuesto antineoplásico taxol y los taxanos relacionados, la producción biotecnológica del cual se basa en estudios empíricos. El objetivo de este trabajo ha sido estudiar como los factores que incrementan la producción de taxanos afectan los perfiles metabólicos y la expresión génica en los cultivos. De esta manera se han identificado nuevos genes candidatos que codifican para los genes desconocidos de la biosíntesis, algunos pasos limitantes de ésta y se ha caracterizado un regulador relacionado con el metabolismo secundario. Se ha ensayado la coronatina, un elicitor más eficiente para mejorar la producción de taxanos y menos dañino que el jasmonato de metilo. En los diferentes ensayos de este trabajo han sido determinados los niveles de expresión de genes que participan en la biosíntesis de taxol, ayudando a comprender su papel en la producción de este anticancerígeno.