Browsing by Author "Pilon, Marinus, committee member"
Now showing 1 - 16 of 16
Results Per Page
Sort Options
Item Open Access Ecological aspects of plant selenium hyperaccumulation: effects of selenium hyperaccumulation on plant-plant interactions(Colorado State University. Libraries, 2016) Mehdawi, Ali Farag El, author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Paschke, Mark, committee member; Vivanco, Jorge, committee memberHyperaccumulators are plants that accumulate toxic elements to extraordinary levels. Selenium (Se) hyperaccumulators such as Astragalus bisulcatus and Stanleya pinnata can contain 0.1-1.5% of their dry weight in Se (1,000 - 15,000 mg Se kg-1 DW), levels toxic to most other organisms. Selenium promotes hyperaccumulator growth and also offers the plant several ecological advantages through negative effects on Se-sensitive partners. Previous work has shown that high tissue Se levels reduce herbivory and pathogen infection. On the other hand, hyperaccumulators may offer an exclusive niche for Se-tolerant ecological partners. The focus of this dissertation study was on the effects of Se hyperaccumulation on plant-plant interactions. The first Chapter presents a literature review of the phenomenon of Se hyperaccumulation, how Se hyperaccumulators are different from other plants, and an overview of previous studies on the effects of hyperaccumulated Se on ecological processes related to herbivore-plant interactions, microbe-plant interactions and pollinator-plant interactions. In addition, evolutionary aspects of Se hyperaccumulation are discussed, and their implications for their ecological partners. The findings presented in this overview formed the platform for the experiments carried out in this dissertation research, on the topic of plant-plant interactions. In Chapter 2, experiments are described to address the question whether Se hyperaccumulation can negatively affect neighboring plants. Soil collected around hyperaccumulators on a seleniferous field site was measured and shown to contain more Se (up to 266 mg Se kg-1) than soil around non-hyperaccumulators. Vegetative ground cover was somewhat lower around Se hyperaccumulators compared to non-hyperaccumulators. Thus, Se hyperaccumulators may increase surrounding soil Se concentration (phytoenrichment). The enhanced soil Se levels around hyperaccumulators were shown to impair growth of a Se-sensitive plant species, Arabidopsis thaliana, pointing to a possible role of Se hyperaccumulation in elemental allelopathy. In Chapter 3, potential positive effects of hyperaccumulator Se on neighboring plants are explored. It was found for two plant species, Artemisia ludoviciana and Symphyotrichum ericoides, that growing next to Se hyperaccumulators increased their Se content 10-20 fold (up to 800-2,000 mg Se kg-1 DW) compared to when they were growing next to non-accumulators. Moreover, these neighbors of hyperaccumulators were 2-fold bigger, showed 2-fold less herbivory damage and harbored 3-4 fold fewer arthropods than when growing next to non-hyperaccumulators. When used in laboratory choice and non-choice grasshopper herbivory experiments, Se-rich neighbors of hyperaccumulators experienced less herbivory and caused higher grasshopper Se accumulation (10-fold) and mortality (4-fold). These results suggest that Se hyperaccumulators can facilitate the growth of Se-tolerant neighboring plants. The fourth Chapter describes a more controlled greenhouse pot cocultivation study that investigated how Se affects relationships between Se hyperaccumulators (A. bisulcatus and S. pinnata) and related non-accumulator species (A. drummondii and S. elata), in terms of how these plants influence their neighbor’s Se accumulation and growth. Selenium affected growth differently in hyperaccumulators and nonaccumulators: The hyperaccumulators performed 2.5-fold better on seleniferous than non-seleniferous soil, and grew up to 4-fold better with increasing Se supply, while the non-accumulators showed opposite results. Both hyperaccumulators and non-accumulators could affect growth (up to 3-fold) and Se accumulation (up to 6-fold) of neighboring plants. The mechanisms for these effects are largely unknown but may involve concentration of soil Se via exudation, root turnover and litter deposition. Exudate of selenate-supplied A. bisulcatus was shown by x-ray absorption spectroscopy to contain mainly C-Se-C. In conclusion, Se hyperaccumulators may enhance the soil Se levels under their canopy, and also convert inorganic Se to organic Se. The Se-enriched soil around hyperaccumulators enhances Se levels in neighboring plants, which may negatively affect Se-sensitive neighboring plants via toxicity, but facilitate Se-tolerant neighbors through reduced herbivory. The latter is an interesting finding, as it constitutes facilitation via enrichment with a non-essential element. It is also interesting that Se enrichment of neighbors by hyperaccumulators can result in competition when neighbors are Se-sensitive and in facilitation when neighbors are Se-tolerant. Via these competitive and facilitating effects, Se hyperaccumulators may affect plant species composition and, consequently, higher trophic levels. Hyperaccumulators may favor Se resistant species at different trophic levels, while selecting against Se sensitive species. If indeed Se hyperaccumulators affect soil Se distribution and speciation and local species composition and Se tolerance, Se hyperaccumulators may play an important role in Se entry into and Se cycling through their seleniferous ecosystems.Item Open Access Functional analysis of three Arabidopsis SR proteins (SCL33, SC35, SCL30A) in plant development and splicing(Colorado State University. Libraries, 2012) Thomas, Julie, author; Reddy, A. S. N., advisor; Bedinger, Pat, committee member; Pilon, Marinus, committee member; Wilusz, Jeff, committee memberTo view the abstract, please see the full text of the document.Item Embargo Mapping the metabolic protein interactome that supports energy conservation at the limits of life(Colorado State University. Libraries, 2024) Williams, Seré Anne, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Pilon, Marinus, committee member; Anderson, G. Brooke, committee member; Snow, Christopher, committee memberDistinct metabolic strategies yield energetic gains from a wide variety of substrates, yet only three overarching methods of energy conservation have been defined: substrate level phosphorylation, the generation of a charged membrane, and electron bifurcation. The dominant theme of known energy conservation mechanisms suggests that energy is conserved through the selective movement and management of electrons, thus essentially all life relies on redox (reduction and oxidation) reactions. Small molecule redox cofactors (such as NAD(P)+) and proteinaceous electron carriers (such as ferredoxins) are employed as electron carriers throughout the biosphere. Proteinaceous electron carriers offer the potential for selective protein-protein interactions to bridge reductive flow from catabolic reactions to the membrane, providing a "proteinaceous electron highway" for efficient electron shuttling. Specific redox protein partnerships have been shown to adapt to changing physiological conditions, suggesting that proteinaceous electron flux is tunable and provides a level of selectivity not possible with small molecule electron transport. While electron flux through a tunable and regulated system of protein interactions can offer exceptional energy conservation strategies, large gaps remain in our knowledge of how electron flux is regulated in vivo. Identification of bona fide in vivo protein assemblies – and how such assemblies dictate the totality of electron flow and thus cellular metabolism – is an important milestone to understand the regulation imposed on metabolism, energy-production, and energy conservation. Resolving the dynamic nature of nanoscale interactions in living systems is arguably the current frontier of molecular biology, and combinatorial methods – which layer multiple in vitro and in vivo techniques with large data analysis – have come to the forefront. This dissertation addresses energy conservation strategies of in vivo protein associations in a model, genetically accessible, hyperthermophilic archaeon (Thermococcus kodakarensis) by mapping the metabolic protein interactome using affinity purification mass spectrometry (AP-MS) and generating engineered strains where fusion proteins selectively redirect electron flux in vivo. Twenty-five proteins involved in distinct metabolic functions were tagged to reveal that each tagged-protein interacts with ~ thirty proteins on average. These interactions connected disparate functions suggesting catabolic and anabolic activities may occur in concert -- in temporal and spatial proximity in vivo. The AP-MS method also refined our understanding of previously determined stable complexes suggesting that protein complexes in vivo likely adapt to redox conditions. Engineered strains linking a proteinaceous electron donor to a proposed electron acceptor were viable and impacted electron flux in vivo. Fusion strains linking a ferredoxin to the hydrogen-generating respiratory system increased hydrogen gas output ~8% on average with one strain showing a ~45% increase over wild type. Fusion strains impacting lipid saturation were shown to inhibit saturation, and future studies aim to determine if electrons can be redirected from the vast reductant sink of lipids to the generation of hydrogen gas, a valuable biofuel.Item Open Access Photoprotection and chloroplast regulation in the green algae Chlamydomonas reinhardtii(Colorado State University. Libraries, 2019) Cantrell, Michael, author; Peers, Graham, advisor; Pilon, Marinus, committee member; Reddy, A. S. N., committee member; Peebles, Christie, committee memberAbsorbed light energy in excess of a cell's photosynthetic capacity can lead to production of reactive oxygen species (ROS) causing cell damage and death. Plants and algae have evolved conserved photoprotective responses that, at the level of light harvesting, are collectively measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence. The major components of NPQ are thermal dissipation of excess light energy (excitation dependent quenching, qE), the migration of antenna complexes from PSII to PSI (state transitions, qT) and inactivation of PSII by damage (photoinhibition, qI). Excess reductant generated during light harvesting can also be dissipated by auxiliary electron transport (AET). The following dissertation aimed to characterize the role of qE in acclimation to saturating and sinusoidal light regimes in the model green algae Chlamydomonas reinhardtii, to characterize potential energy dissipating mechanisms that may occur in absence of qE and identify factors regulating the expression of the chloroplast encoded photosystem I subunit, psaA, using a forward genetic screen. In chapter 2 I show that the qE mutant, npq4lhcsr1, displays decreased growth under a sinusoidal light regime mimicking natural oscillations in irradiance. This reduction in growth rate occurs without a significant impact on carbon accumulation, accumulation of oxidized lipids or impairment of photosynthetic rate. We hypothesized that this was due to increased consumption of excess energy by AET pathways and the results of this investigation are presented in chapter 3. We found that absence of qE in Chlamydomonas did not significantly impact AET associated with light dependent oxygen consumption. The npq4lhcsr1 mutant instead appears to experience less acceptor side limitation downstream of Photosystem I and have a greater capacity for state transitions. This in the absence of any evidence for increased light dependent oxygen consumption in the npq4lhcsr1 mutant indicates that Chlamydomonas compensate for the absence of qE by increasing cyclic electron transport around Photosystem I, which generates additional ATP at the cost of NADPH. In my final chapter I use a positive selectable marker to generate a library of 400 putative psaA mutants, present preliminary flanking sequence characterization for 29 of these mutants and discuss possible roles they may be playing in psaA regulation. Together these chapters expand our understating of the role of qE in long term acclimation to saturating and sinusoidal light regimes and provide a library of putative chloroplast regulatory mutants that, with further characterization, will refine our understanding of chloroplast genome regulation in green algae.Item Open Access Photosynthesis in dynamic and rapidly changing light: the physiology of a cyanobacterium in a photobioreactor(Colorado State University. Libraries, 2017) Andersson, Bjoern, author; Peers, Graham, advisor; Pilon, Marinus, committee member; Peebles, Christie, committee memberMass cultivation of aquatic phototrophs in photobioreactors (PBRs) has the potential to produce sustainable biofuels thus reducing net carbon emission and associated climate change. In order to make PBRs productive enough to be economically viable, the biomass accumulation rate and cell density at harvest needs to be high. However, early productivity estimates based on controlled laboratory experiments has not scaled-up to industrial size PBRs. One major reason is that the growth rates in high density, low maintenance PBRs is severely reduced compared to laboratory conditions. This is likely a consequence of the fluctuating light environment. The photophysiological response of algae or cyanobacteria to growth in outdoor PBRs has not been well characterized. The work presented in this thesis aimed to describe the complexity of the light environment in a small-scale PBR and also the physiological response of photoautotrophs to growth in this environment. A dense culture of the cyanobacterium Synechocystis sp. PCC 6803 was grown in a bench-top PBR with an incident light that followed a sinusoidal function peaking at 2000 µmol photons m-2 s-1. These conditions approximate natural sunlight. The diurnal changes in the light environment of the bench top PBR was quantified from the perspective of a single-cell, using a computational fluid dynamic approach (Chapter 1). Due to self-shading within the dense culture, single cells experienced rapid fluctuations (~6 s) between 2000 and <1 µmol photons m-2 s-1, and on average the integrated irradiance per cell was 85% lower than the incident irradiance (mean per cell: 184 µmol photons m-2 s-1). We investigated the activity of photoprotective mechanisms under our realistic light environment, using pulse amplitude modulated (PAM) fluorometery and membrane inlet mass spectrometry (MIMS). Contrary to common assumption we found no evidence for net-photodamage or non-photochemical quenching (NPQ) activity in situ (Chapter 1). In an ex situ experiment we found that alternative electron transport (AET) dissipated 50% of electrons from photosystem II, preventing them from being used for carbon fixation. This indicates that AET, and not NPQ is the first photoprotective mechanism Synechocystis uses under dynamic and fluctuating light. These results have important applications for genetic and metabolic engineering strategies that commonly targets NPQ and photodamage as a way to boost productivity of PBRs. Since, AET caused the main diversion from linear electron transport and carbon fixation, this mechanism should be investigated as a genetic engineering strategy. Samples were also taken to monitor the response of the transcriptome with high temporal precision around the day/night transitions (Chapter 2). The transcriptome data showed that 74% of all genes exhibited some modification in transcription across the diel cycle. In my preliminary analysis of the data (Chapter 2), I found that the major components of photosynthetic light harvesting and electron transport complexes increased in abundance during the whole light period. This is commonly observed in cultures growth under sub-saturating light intensities but not high light stress. Furthermore, few other high light stress responses were observed in the transcriptome. There was little diel variation in transcriptional activity of molecular chaperones (dnaK, hsp, groE families), proteases (ftsH and Deg families), high light inducible proteins (hli), and reactive oxygen species scavengers (superoxide dismutase and catalase peroxidase) that are responsive to high light stress. The flavodiiron proteins are considered the main player of AET in cyanobacteria and are up-regulated transcriptionally under light and inorganic carbon stress. Interestingly, there were no increased abundance in transcripts of the flavodiiron proteins during the light period in my experiment. Assuming that transcript abundance correlates with protein abundance this could mean that either these genes are constituently expressed or that other enzymes may exist that are responsible for the AET. Further analysis of the transcriptomic data and future proteomic analysis may uncover putative genes whose transcriptional pattern indicates that they may play a role in AET under fluctuating light.Item Open Access Phylogenetic and population genetic evidence for positive selection in rapidly evolving plastid-nuclear enzyme complexes(Colorado State University. Libraries, 2016) Rockenbach, Kate, author; Sloan, Daniel, advisor; Argueso, Cris, committee member; Pilon, Marinus, committee member; Mueller, Rachel, committee memberRates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid-nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic co-evolution between pathogens and host immune genes. We discuss a possible role of plastid-nuclear conflict as a novel cause of accelerated evolution.Item Open Access Physiological response of the Cyanobacterium synechocystis sp. PCC 6803 to fluctuating light(Colorado State University. Libraries, 2015) Youngblood, Matthew Thomas, author; Peers, Graham, advisor; Peebles, Christie, committee member; Pilon, Marinus, committee memberPhotosynthetic microbes are a promising feedstock for renewable biofuels, but the yields of industrial cultivation systems will need significant improvements if they are to be economically viable and succeed. One particular challenge faced by photosynthetic microbes in commercial production systems is the highly dynamic light environment created by vertical mixing within dense cultures. Rapid changes in light intensity make it difficult for these microbes to acclimate and utilize the available light efficiently. Attempts to identify targets for genetically improving photosynthetic microbes to flourish in these environments are hampered by a poor understanding of the physiological response to fluctuating light. My thesis is focused on developing our fundamental understanding of the photophysiology and acclimation responses associated with light environments in industrial conditions. The aim is to eventually increase areal productivity in industrial cultivation systems by applying insights from physiological characterization into future strain engineering approaches. The first chapter introduces the issues associated with industrial cultivation of photosynthetic microbes. I present some background on the need for biofuels, why there has been a focus on using photosynthetic microbes as a feedstock, and how photosynthetic microbes are cultivated industrially. I then present some of the physiological challenges faced by photosynthetic organisms in industrial cultivation, with particular focus on the challenge of a dynamic light environments. I give a brief background on photosynthesis to explain some of the acclimation responses that can be altered in a fluctuating light environment. Chapter 2 presents a proteomic and physiological comparison of the cyanobacterium Synechocystis sp. PCC6803 cultivated in a fluctuating light environment (30s light on/off) to a continuous light environment which had the same average photon flux density. We found that cultures in fluctuating light grew at half the exponential growth rate of continuous light cultures. Reduced growth did not appear to be due to photo-oxidative stress, as we detected reduced levels of reactive oxygen species and oxidative-stress responsive proteins in fluctuating light. We show evidence that reduced growth could be due to a partial shift to a respiratory state in fluctuating light. Reduced growth could also be due to increased dissipation of electrons, as suggested by the higher capacity for photosynthesis at light levels from 106-174 μmol photon m⁻² s⁻¹. We found other surprising changes such as increases in some carbon concentration mechanism components and decreases in some others. These components were thought to be regulated by a similar mechanism due to their co-expression in high CO₂ to ambient CO₂ shift experiments. This suggesting some unusual signaling is occurring due to the fluctuating light. We also found a number of hypothetical and poorly characterized proteins were significantly different in fluctuating and continuous light. In the appendix, I present the preliminary characterization of slr1719, a poorly characterized protein identified in the proteomic analysis of fluctuating and continuous light. This study generated a knock out of slr1719. We found that the Δslr1719 strain compared to a control strain grew much slower in low CO₂ conditions in saturating and sub saturating light conditions. However in replete CO₂ conditions, there was no difference in growth rate, regardless of light intensity tested in Δslr1719 versus the control. We argue that this finding, paired with evidence from the literature, suggests slr1719 may participate in cyclic electron flow. Other roles suggested by the literature for this protein could have important roles in the acclimation to fluctuating light, and warrant further study.Item Open Access Phytoremediation and biofortification potential of Cannabis sativa L.(Colorado State University. Libraries, 2019) Stonehouse, Gavin C., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Ippolito, Jim, committee memberSelenium (Se) is a micronutrient, but toxic at high levels. Both Se deficiency and toxicity are problems worldwide. I studied the potential of hemp (Cannabis sativa L.) for Se environmental cleanup (phytoremediation) and for accumulating elevated levels of this healthy micronutrient (Se biofortification). Hemp properties attractive for phytoremediation are fast growth, high biomass, hardiness and economic value. Furthermore, hemp produces highly nutritious seeds, of interest for Se biofortification. The first Chapter of this thesis reviews Cannabis sativa's history, biological attributes and applications, as well as the technologies of phytoremediation and biofortification, and plant Se metabolism. The second Chapter presents experimental data on two hemp studies. The first was a field survey of Se accumulation in hemp grown across Colorado, and in commercial hemp products. The second study involved controlled greenhouse experiments to study hemp Se tolerance, accumulation and metabolism. Hemp field surveys in four naturally seleniferous (Se-rich) agricultural areas in Colorado, U.S.A. found 15-25 µg Se/g in seed (intact or dehulled) and 5-10 µg Se/g dry weight in flowers and leaves. Hemp beer contained 42 µg Se/L. Considering the U. S. recommended daily allowance (RDA) of 55-75 ug Se, one bottle of hemp beer provides 25%, and 4 gram hemp seed (a half tablespoon) provides 100% of the RDA. In controlled greenhouse experiments, hemp was further characterized for Se tolerance, accumulation and Se speciation. Effects of Se on photosynthesis and cannabinoid and terpenoid levels were also analyzed. At the seedling level, hemp showed high selenate tolerance (up to 160 µM) and accumulation (up to 1,400 mg Se/kg shoot dry weight). Mature hemp was completely tolerant up to 40 µM selenate and accumulated up to 200 mg Se/kg DW in leaves, flowers and seeds. Seeds were found to contain free (water-extractable) selenomethionine and methyl-selenocysteine, superior forms for Se biofortification, reported to have anticarcinogenic properties for consumers. Hemp production of medicinal cannabidiol (CBD) and terpenoids was not affected by Se. Selenium enhanced potassium levels in seeds, and thus their nutritional value; other nutrient levels were unaffected. It can be concluded from these studies that hemp shows promise for Se phytoremediation and can produce Se-biofortified dietary products; Se does not affect levels of valuable secondary plant compounds, nor does it negatively affect nutritional quality of seeds. These findings are of significance in view of the widespread and rapidly expanding cultivation of hemp in seleniferous areas across the U.S.A. and Canada.Item Open Access Phytoremediation with hemp (Cannabis sativa L.): a look at hemp's potential for environmental cleanup and economic recovery(Colorado State University. Libraries, 2022) Abernathy, Susan M., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Qian, Yaling, committee memberThe aim of this thesis study was to test hemp's (Cannabis sativa L.) potential for phytoremediation (environmental clean-up). I tested hemp for tolerance and accumulation of four inorganic pollutants, to evaluate its remediating performance. Hemp has many properties that would make it a likely candidate for phytoremediation however, due to recent regulations, research of this versatile plant has been limited. Phytoremediation is a process of cleaning polluted sites using plants. In this clean-up method, plants may stabilize the pollutant in situ, or take-up the pollutant into the plant tissue. In the latter, there are a few different fates for the pollutant that include degradation, metabolization, sequestration, and/or volatilization. Phytoremediation is a clean process that reestablishes an onsite ecosystem and is a competitive alternative to more conventional scrape-and-remove methods. Hemp is a hardy, fast growing species that produces high biomass. Hemp has deep roots that can be used to reach pollutants deep in the ground. These properties make hemp a potential choice for phytoremediation. Contaminated sites create harsh growing conditions that require hardy plant properties in order for a species to survive. An added benefit to using hemp for remediation is the many economic uses of hemp biomass. Each part of the hemp plant can be used to make goods such as clothing, building material, cosmetics, lotions, animal bedding, fragrances, and medicinal products that have therapeutic qualities. In addition, hemp seeds are nutritious and can be added to the diet. In chapter one of this thesis, phytoremediation is reviewed to explain the remediation process. This review includes explaining the different phytotechnologies that are employed by plants which depend on the plant used and the type of pollutant encountered. Chapter one also reviews hemp, its history, biology, and the properties that make it a viable choice for phytoremediation. Chapter two of this thesis is an experimental chapter presenting data for testing hemp seedlings with four different oxyanions: arsenate (As), molybdate (Mo), vanadate (V), and tungstate (W). The parameters considered were biomass, chlorophyll content, chlorophyll fluorescence, pollutant accumulation levels, and pollutant fate. Brassica juncea (Indian mustard) was used as a reference phytoremediation species. The findings of this thesis study present promising results for hemp as a potential remediator. Arsenic was found to accumulate in the root at levels up to 2700 mg kg-1 DW. Tungsten also accumulated in the root at levels up to 3100 mg kg-1 DW. In both tests, hemp performed well, judged from photosynthetic measurements and relative chlorophyll content, but reduced biomass started at treatments with 3 and 24 mg As L-1 in the shoot and root respectively, and 40 and 80 mg W L-1 in the shoot and root, respectively. Molybdenum accumulated in the shoot at levels up to 4900 mg kg-1 DW and in the root at levels up to 2600 mg kg-1 DW. Biomass reduction of Mo started at treatment with 40 mg Mo L-1 for both shoot and root, while photosynthetic measurements and relative chlorophyll content remained unchanged. Lastly, V accumulated in the root at levels up to 2100 mg V kg-1 DW. Interestingly, hormesis (stimulated growth) was observed in hemp supplied with V: biomass increased at all tested levels. From this study, it was concluded that hemp may have potential for phytoremediation in cleaning contaminated sites with the four elements tested. Hemp performed competitively with the popular phytoremediation species, Indian mustard (Brassica juncea L.) in all levels tested for Mo, V, and W. Hemp's economic recovery with clean post-harvest biomass may offset phytoremediation costs giving this species a unique advantage over other popular phytoremediation choices.Item Open Access Protoporphyrinogen oxidase: origins, functions, and importance as an herbicide target site(Colorado State University. Libraries, 2021) Barker, Abigail, author; Dayan, Franck, advisor; Snow, Christopher, committee member; Pilon, Marinus, committee member; Gaines, Todd, committee memberProtoporphyrinogen IX oxidase (PPO)-inhibiting herbicides are effective tools to control a broad spectrum of weeds, including those that have evolved resistance to glyphosate. Their utility is being threatened by the appearance of biotypes that are resistant to PPO inhibitors. While the chloroplastic PPO1 isoform is thought to be the primary target of PPO herbicides, evolved resistance mechanisms elucidated to date are associated with changes to the mitochondrial PPO2 isoform, suggesting that the importance of PPO2 has been underestimated. Our investigation of the evolutionary and structural biology of plant PPOs provides some insight into the potential reasons why PPO2 is the preferred target for evolution of resistance. The most common target-site mutation imparting resistance involved the deletion of a key glycine codon. The genetic environment that facilitates this deletion is apparently only present in the gene encoding PPO2 in a few species. Additionally, both species with this mutation (Amaranthus tuberculatus and Amaranthus palmeri) have dual targeting of PPO2 to both the chloroplast and the mitochondria, which might be a prerequisite to impart herbicide resistance. The most recent target-site mutations have substituted a key arginine residue involved in stabilizing the substrate in the catalytic domain of PPO2. This arginine is highly conserved across all plant PPOs, suggesting that its substitution could be equally likely on PPO1 and PPO2, yet it has only occurred on PPO2, underscoring the importance of this isoform for the evolution of herbicide resistance. As glyphosate resistance becomes widespread, weed control turns to older mechanisms of action with less resistance. Protoporphyrinogen oxidase (PPO) inhibitors are a versatile class of herbicides that have been used since the 1960's, with active ingredients that work in pre-emergent and post-emergent applications. Differential efficacy of PPO inhibitors applied pre-emergent, early post-emergent and late post-emergent has been observed in multiple species and settings. Understanding the cause of higher efficacy in younger plants could preserve these important weed control tools. To understand the differing efficacies elements that affect the mechanism of action of PPO inhibitors were analyzed over the course of plant growth including target site transcript levels and protein levels, herbicide uptake, antioxidant capacity, and indicators of flux through the pathway. Data show levels of PPO do not explain differential efficacy. Increases of glutamate, the pathway precursor, do increase damage due to PPO inhibitor treatment, but increased levels are not observed in younger plants. Differential efficacy is likely due to a combination of increase in antioxidant capacity and a decrease in herbicide uptake. Other possible factors such as metabolism will need to be measured in future work. Protoporphyrinogen oxidase (PPO) is a critical enzyme across life as the last common step in the synthesis of many metalloporphyrins. The reaction mechanism of PPO was assessed in silico and the unstructured loop near the binding pocket was investigated. The substrate, intermediates, and product were docked in the catalytic domain of PPO using a modified Autodock method, introducing flexibility in the macrocycles. Sixteen PPO protein sequences across phyla were aligned and analyzed with Phyre2 and ProteinPredict to study the unstructured loop from residue 204–210 in the H. sapiens structure. Docking of the substrate, intermediates, and product all resulted in negative binding energies, though the substrate had a lower energy than the others by 40%. The α-H of C10 was found to be 1.4 angstroms closer to FAD than the β-H, explaining previous reports of the reaction occurring on the meso face of the substrate. A lack of homology in sequence or length in the unstructured loop indicates a lack of function for the protein reaction. This docking study supports a reaction mechanism proposed previously whereby all hydride abstractions occur on the C10 of the tetrapyrrole followed by tautomeric rearrangement to prepare the intermediate for the next reaction. Weed control is essential in modern agriculture, though it becomes more difficult with increasing resistance levels to current herbicides and a slow process to register a new mechanisms of action because of safety concerns and current methods. Agrematch provides a new method to identify possible herbicide candidates using an artificial intelligence algorithm that takes into effect biological parameters with the goal of reducing R&D time on new herbicides. Herein we describe the discovery of 4-chloro-2-pentenamides as novel inhibitors of protoporphyrinogen oxidase, a known herbicide target site, by the Agrematch AI. The herbicidal activity is confirmed in greenhouse assays, with the highest performing AGR001 showing good activity pre-emergent at 150 g/ha and post emergent as low as 50 g/ha on the troublesome weed palmer amaranth (Amaranthus palmeri). A lack of activity is shown on PPO resistant palmer amaranth carrying the ΔG210 deletion mutation. The mechanism of action is confirmed by the herbicide dependent accumulation of protoporphyrin IX, subsequent light dependent loss of membrane integrity, and direct inhibition of protoporphyrinogen oxidase in an in vitro assay. Modeling of the docking of these inhibitors in the active site of protoporphyrinogen oxidase confirms the target.Item Open Access Selenium accumulation in plants and implications for human health: a survey of molecular, biochemical, and ecological cues(Colorado State University. Libraries, 2022) Lima, Leonardo Warzea, author; Pilon-Smits, Elizabeth, advisor; Schiavon, Michela, committee member; Pilon, Marinus, committee member; Antunes, Mauricio, committee member; Paschke, Mark, committee memberTo view the abstract, please see the full text of the document.Item Open Access Selenium uptake, differentiation and metabolism in hyperaccumulator Stanleya pinnata(Colorado State University. Libraries, 2013) Harris, Jonathan, author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Peers, Graham, committee member; Ward, Sarah, committee memberSelenium (Se) is a biologically essential element for many animals, some prokaryotes and algae. However, even in organisms that require Se, the range between sufficiency and toxicity for Se is narrow. Although there are no reports of a Se requirement or selenoproteins in higher plants, there are species that appear endemic to seleniferous soil and concentrate Se in their leaves to levels exceeding 1000 mg kg-1 dry weight. These plants are known as Se hyperaccumulators and have an exceptional ability to tolerate and enrich themselves with this toxic element. As a result of the Se concentrations in their tissues, Se hyperaccumulators are extremely toxic to most organisms. Studies have found that Se hyperaccumulation protects these plants from many herbivores and pathogens as an "elemental defense." Some of these hyperaccumulators have been studied for their use in phytoremediation of naturally occurring and anthropogenically contaminated seleniferous soils. Although the slow growth of most hyperaccumulators limits their direct application for phytoremediation, they can be utilized as a source of genes to genetically enhance Se accumulation and tolerance in popular phytoremediator species. The goal of this study is to better characterize the uptake, metabolic fate and molecular mechanisms responsible for Se tolerance in Stanleya pinnata, a hyperaccumulator in the Brassicacae. Two main techniques were utilized: physiological experiments followed by elemental analysis to characterize Se uptake and interactions with the related element sulfur (S), and Illumina sequencing of the transcriptomes of Stanleya pinnata and related non-hyperaccumulator Stanleya elata. The first chapter presents a literature review of Se hyperaccumulation: what is known about Se assimilation in higher plants, and some unique characteristics of hyperaccumulators. The metabolism of Se through the sulfate assimilation pathway is described, and known mechanisms of Se tolerance and accumulation in representative plants are reviewed. In addition, some of the previous work on Stanleya is reviewed, including a number of studies that have shown ecological benefits of Se hyperaccumulation. Known beneficial genes for Se tolerance and accumulation are discussed in the context of phytoremediation. In chapter 2, Se-specific uptake was tested in two ecotypes of S. pinnata, and contrasted with related non-hyperaccumulator Brassica juncea. To test for Se specificity of sulfate transporters, plants were supplied with varying concentrations of selenate and two concentrations of sulfate. The results showed that S. pinnata is able to take up large amounts of Se, even at exceedingly low supplied Se:S ratios. In addition, S. pinnata preferentially mobilized large amounts of Se to young leaves, without commensurate mobilization of S. These trends were not observed in the non-hyperaccumulator B. juncea, which showed dramatically reduced Se uptake under elevated sulfate supply. Moreover, there was no evidence of preferential allocation of Se to young tissues in B. juncea. Taken together, these findings support the hypothesis that Stanleya contains transporters with an increased specificity for Se, allowing it to take up preferentially and mobilize Se over S. Since previous work has shown that molybdate may be taken up in part by plant sulfate transporters, this element was also monitored. It was observed that increasing supply of selenate and sulfate significantly reduced the molybdenum (Mo) content of leaves in S. pinnata. In contrast, B. juncea showed an increase in Mo content with increases in supplied selenate. In the experiment described in Chapter 3, Illumina sequencing was performed to compare the root and shoot transcriptomes of hyperaccumulator S. pinnata and non-hyperaccumulator S. elata in the presence or absence of selenate. An overview is presented of the overall transcriptome response patterns, followed by a more detailed analysis of transcripts involved in S/Se metabolism. In the presence of Se, 40 of the 56 S/Se-related genes were more highly expressed in S. pinnata than S. elata. Particularly promising findings include a vastly upregulated root sulfate/selenate transporter (Sultr1;2) and ATP sulfurylase (APS2). Lastly, some preliminary findings are presented from several biochemical approaches used to further investigate S. pinnata hyperaccumulation mechanisms. Organic forms of Se were investigated in S. pinnata and S. elata using a newly developed liquid chromatography mass spectrometry (LC-MS) method. It was shown that S. pinnata accumulates significant amounts of selenocystathionine as well as methyl-selenocysteine. Moreover, activities of selenocysteine lyase (SL) and cysteine desulfurase (CysD) were investigated in S. pinnata and S. elata, which revealed strong SL activity in the hyperaccumulator. The possible role of this enzyme in Se hyperaccumulation remains to be elucidated. Finally, superoxide dismutase activities were compared between the two species in relation to Se supply. Stanleya pinnata and other Se hyperaccumulators may be valuable resources for genes involved in Se tolerance and hyperaccumulation, to create genetically engineered plants for phytoremediation purposes. In addition to the potential environmental benefits, understanding potential biological roles for Se and its metabolism in these plants may have broad applications for human health. Many organic seleno-compounds have been studied for their anti-carcinogenic properties in multiple systems and types of cancer. Efficacy of these Se compounds appears to vary based on the form of Se. Plants capable of creating different forms of organic Se may become a valuable pharmaceutical resource.Item Open Access Studies on selenium hyperaccumulator Stanleya pinnata and nonaccumulator Stanleya elata (Brassicaceae): functional characterization of selenate transporter SULTR1;2 in yeast and development of a micropropagation protocol(Colorado State University. Libraries, 2017) Guignardi, Zackary S., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Santangelo, Thomas, committee memberStanleya pinnata is an herbaceous perennial species in the family Brassicaceae native to the western United States. This species is classified as a selenium (Se) hyperaccumulator, and can be found thriving on Se-rich soils. Selenium hyperaccumulators are plant species that have the capacity to accumulate Se over 1,000 mg kg-1 dry weight in their tissues, concentrations toxic to non-accumulator plant species as well as to herbivores and pathogens, which may explain why plants hyperaccumulate Se. Due to the chemical similarity of Se to sulfur (S), Se is believed to be transported and metabolized by the same proteins and enzymes, including sulfate transporters and the sulfate assimilation pathway. Selenate (SeO42-), the predominant available form of Se in soil, is transported into the roots mainly via the high-affinity membrane transporter SULTR1;2. While most plants do not appear to discriminate between selenate and sulfate, and the two compounds compete for uptake, selenate uptake in Se hyperaccumulators is less inhibited by high sulfate concentrations. Since SULTR1;2 is the main portal of entry for selenate into the plant, it may be hypothesized that SULTR1;2 from the Se hyperaccumulator S. pinnata has intrinsic properties that allow this species to discriminate between sulfate and selenate and preferentially take up selenate. One of the objectives of this thesis project was to test this hypothesis, by means of functional characterization of SULTR1;2 from S. pinnata and from control species Stanleya elata, and Arabidopsis thaliana in the YSD1 yeast mutant which lacks its native sulfate transporters. A secondary objective in this thesis project was to develop a micropropagation protocol for Stanleya. In order to effectively study Se hyperaccumulation in a laboratory setting, sufficient numbers of S. pinnata and S. elata plants need to be available. However, due to low rates of seed germination, vernalization requirements, self-incompatibility, and ineffectiveness of propagation by cuttings, conventional propagation methods via seed or vegetative cuttings severely limit the number of plants that can be cultivated at a time. In order to overcome these limits, a tissue culture micropropagation protocol for leaf explants of S. pinnata and S. elata was developed. This protocol will allow for the rapid reproduction of both Stanleya species, not only to be used in laboratory experiments, but also in industrial applications such as Se phytoremediation projects, as well as for horticultural and native landscaping purposes. The first chapter of this thesis reviews plant Se uptake and metabolism, offering an overview of the current understanding of the Se assimilation pathway in plants, including mechanisms of accumulation and tolerance unique to Se hyperaccumulators. This chapter also outlines key proteins and enzymes in the Se assimilation pathway that are candidates for future experiments to determine the mechanisms of Se hyperaccumulation. The second chapter describes the results from yeast studies, characterizing the selenate and sulfate transport capabilities of SULTR1;2 from hyperaccumulator S. pinnata and non-accumulators S. elata, and A. thaliana, and their selenate specificity, as judged from the effects of sulfate competition on selenate uptake. Interestingly, yeast transformed with SULTR1;2 from S. pinnata (SpSultr1;2) showed less inhibition of selenate uptake by high sulfate concentration, indicating that this species' selenate selectivity may be facilitated by the SULTR1;2 protein. While apparently more Se-specific, yeast transformed with SpSultr1;2 overall took up less Se when compared to yeast expression SULTR1;2 from non-accumulators. It is feasible that a mutation that changes the substrate specificity of SpSULTR1;2 also reduced its overall activity. In S. pinnata, SpSultr1;2 transcript was found in earlier studies to be ~10-fold up-regulated when compared to S. elata, which may compensate for decreased activity. Identification of a selenate-specific transporter has applications for Se phytoremediation and biofortification. Constitutive overexpression of a hyperaccumulator selenate transporter in other plant species may increase their uptake of Se, even in the presence of high environmental S levels. The third chapter of this thesis outlines the development of a fast and efficient tissue culture micropropagation protocol for S. pinnata and S. elata. Through the testing of multiple concentrations of hormones on in vitro callus formation, shoot induction and elongation, and root formation, followed by ex vitro acclimatization, both species of Stanleya were shown to be very amenable to micropropagation. Both exhibited rapid callus, shoot, and root induction under a wide range of 1-napthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), and indole-3-butyric acid (IBA) concentrations. Future experiments could explore the genetic transformation of S. elata plants with genes from S. pinnata to test their importance for Se accumulation and tolerance in this related non-accumulating species. This micropropagation protocol also opens up the possibility to cultivate the Stanleya species at a large scale for multiple applications including biofortification, phytoremediation, and native landscaping.Item Open Access The evaluation of the potential for chlorine dioxide to prime plant defenses for a systemic acquired resistance in light red kidney bean plants inoculated with common bean bacterial wilt(Colorado State University. Libraries, 2015) Sandoval, Vanessa Marie, author; Newman, Steven, advisor; Ramsey, Craig, advisor; Pilon, Marinus, committee member; Qian, Yaling, committee memberThe induction of plant defenses is a great preventative tool for greenhouse and nursery managers to protect their plants. By priming plants with abiotic or biotic measures, managers can induce systemic acquired resistance (SAR) in plants to upregulate the ability to resist a pathogen. The accumulation of salicylic acid (SA) has been well researched and supported to be necessary for inducing SAR against pathogens. In previous research it has been shown that the functional analog of SA, acibenzolar S-methyl, has induced SAR and reduced disease severity. Acibenzolar S-methyl induces SAR when applied to plant foliage, but it does not have any antimicrobial activity to kill any pathogens on the foliage at the time of treatment. In previous research ozone has been successful at inducing SAR to reduce disease severity. Applying ozone as a treatment for greenhouse and nursery managers is not practical or safe since it is hazardous to the respiratory system. Chlorine dioxide is a powerful oxidant disinfectant that can be applied as a foliar spray to kill harmful pathogens, but it has not been reported whether it could induce plant defenses. This research study investigated whether a commercial formulation of chlorine dioxide [Electro-biocide® (E-B)] could be used as a foliar application to plants to induce SAR. E-B is a proprietary blend of ClO2, pH buffer, and a sarcosinate surfactant. There were a total of four spray treatments that were evaluated on plants inoculated with a bacterial wilt and on a set of non-inoculated plants. The light red kidney bean plants were treated with E-B at 200 mg l-1 ClO2, E-B 400 mg l-1 ClO2, acibenzolar S-methyl (Actigard™) and a water control to evaluate disease resistance when inoculated with Curtobacterium flaccumfaciens pv. flaccumfaciens. Treated plants were evaluated for both inoculated plants and non-inoculated plants. SA concentrations were measured five days after treatment and one day after inoculation. Leaf samples were collected to measure SA every three hours over the course of the day starting at 0700 hours and ending 2200 hours. A second SA measurement was taken at the end of the study 61 days after planting (44 days after treatment) to observe if there were any changes in SA level. Chlorophyll fluorescence measurements were taken to observe stress in response to the spray treatments and disease infection. Carbon dioxide (CO2) gas exchange measurements were taken to observe the vigor or decline within the spray treatments and infection status. At the end of the study plants were harvested for foliage, pod, stem dry weight, and leaf area. The first photosynthesis measurements on non-inoculated plants E-B 200 mg ClO2 l-1 and 400 mg l-1 ClO2 treatments declined, but recovered to control levels one week later. Inoculated plants treated with E-B and Actigard™ showed either the same or increased photosynthesis rates when compared to water. Chlorophyll fluorescence measurements indicated there was no stress due to the spray treatments. Five days after spray treatments the SA measurements showed that both concentrations of E-B resulted in an increase in SA accumulation. E-B 400 mg l-1 ClO2 caused the greatest SA response. E-B 400 mg l-1 ClO2 treated plant’s had a 15 fold increase in SA concentrations at its highest peak when compared to water. E-B 200 mg l-1 ClO2 had the second highest SA concentrations. It had a 5.9 fold increase at its highest peak when compared to water control plants. Actigard™ treated plants did not result in different SA concentrations from the water control plants. The SA concentrations levels at 44 days after treatment for all plants that were not inoculated returned to normal levels. SA levels for inoculated plants and all spray treatments continued to rise for the duration of the study. There were no differences in biomass measurements between spray treatments. All non-inoculated plants had a greater biomass measurements when compared to all the inoculated plants. These results conclude that E-B 200 mg l-1 ClO2 and E-B 400 mg l-1 ClO2 were able to prime plant defenses for SAR response. The rise in SA concentrations confirm that E-B was able to interact within the leaf as an elicitor for SAR. Unfortunately the biomass measurements for inoculated E-B treated plants did not show any difference from inoculated control plants. This indicates that the E-B treatment was not able to reduce the disease severity with CFF. Actigard (acibenzolar-S methyl) has been successful with inducing SAR and reducing disease severity in other studies. In this study Actigard was also unsuccessful in reducing disease severity. This indicates that CFF may have had too great of pressure for the inoculated plants to overcome. E-B should be investigated further with other pathogens.Item Open Access The influence of extensin cross-linking on biomass recalcitrance(Colorado State University. Libraries, 2015) Fleming, Margaret Brigham, author; Bedinger, Patricia, advisor; Pilon, Marinus, committee member; Decker, Stephen, committee member; Kassenbrock, C. Kenneth, committee member; Peebles, Christie, committee memberPlant cell walls are under investigation as a source for biofuel production, yet conversion of cell walls (biomass) into biofuel is currently too expensive to be competitive with gasoline. Biomass is recalcitrant; that is, it resists enzymatic degradation by cellulases into monosaccharides such as glucose. One source of recalcitrance may be the presence of extensins, covalently bound cell wall proteins that are extremely insoluble. To determine what influence, if any, extensins have on biomass recalcitrance, I performed several experiments. I first turned to poplar biomass, which is a model source for biofuels. I found that protease treatment of poplar biomass after liquid hot water pretreatment reduced the hydroxyproline content (a proxy for extensins). The reduction in hydroxyproline content correlated with reduced recalcitrance, seen as an increase in glucose release after cellulase digestion of poplar biomass. I also tested whether Arabidopsis T-DNA insertional mutations in the genes encoding enzymes that perform extensin post-translational modifications could reduce extensin content or cross-linking, and whether this reduction was associated with reduced biomass recalcitrance. I found that although these mutants were hypothesized to have reduced incorporation of extensin in cell walls, no significant effects on extensin content in inflorescence stem cell walls (an analog for woody biomass), nor on glucose release from biomass, were found in any mutant line. Finally, I looked at the effects of extensin overexpression on glucose release in transgenic Arabidopsis lines containing synthetic genes encoding the complete extensin domain from SlLRX1 or a short C-terminal region of 20 amino acids of SlLRX1, fused to the red fluorescent reporter protein tdTomato. Observation of the tdTomato fluorescence in transgenic biomass after various chemical and enzymatic treatments indicated that the C-terminal 20 amino acids of SlLRX1 are sufficient to allow a strong association with the cell wall, while the complete SlLRX1 extensin domain leads to an even stronger, perhaps covalent linkage. Lines transformed with the complete SlLRX1 extensin domain had more than twice the hydroxyproline content in their stems than wild-type, but this increase in hydroxyproline did not affect the amount of glucose released from stems upon cellulase digestion. Since protease treatment reduced both hydroxyproline content and recalcitrance in poplar biomass, further experiments to assess the nature of the association between extensins and cell walls are warranted to attempt to further reduce recalcitrance. In the experiments I performed, the stems of extensin modification mutant Arabidopsis lines showed no change in extensin modification, and therefore no effect on recalcitrance was observed; stems of transgenic overexpression Arabidopsis lines showed increased extensin content, but again, no effect on recalcitrance was observed. My investigations in Arabidopsis focused on stem tissue, as this is analogous to material used in biofuel production. However, extensins are most abundantly expressed in roots in many plants, particularly in Arabidopsis. Examination of roots of both mutant and transgenic Arabidopsis may be more revealing of the interactions between extensins, cell walls, and recalcitrance.Item Open Access The plastid caseinolytic protease complex as a model for cytonuclear coevolution(Colorado State University. Libraries, 2021) Williams, Alissa Marie, author; Sloan, Daniel, advisor; Bedinger, Patricia, committee member; Mueller, Rachel, committee member; Pilon, Marinus, committee member; Stenglein, Mark, committee memberCoevolution, or evolution in response to reciprocal selective pressures, is important to biological function and the persistence of populations. Competition or mutualisms between organisms can drive coevolution, as can predatory or parasitic relationships. However, coevolution also occurs within cells, as coevolution can result from the interactions between proteins within complexes as well as between the multiple genomes within eukaryotic cells. In protein complexes, subunits must bind tightly and specifically to one another. Changes in one protein subunit are often correlated with changes in the other subunits to preserve the functionality of the complex. Thus, in many protein complexes, correlated rates of evolution are found between the sequences of component subunits. This covariation is strong enough to be used as a method to predict which proteins are connected physically and/or functionally. The coevolution between multiple genomes in eukaryotic cells is known as cytonuclear coevolution. Plants, for example, have a nuclear genome and two cytoplasmic genomes found in the plastid (chloroplast) and mitochondrion. Many protein complexes within these organelles consist of subunits deriving from both the nucleus and the organelle itself. Since the nuclear genome and organelle genomes differ in modes of transmission, mutation rates, and selective pressures, partnerships between proteins originating from two cellular compartments are great models for understanding protein complex evolution. Protein complexes are frequently shaped via gene duplication. Many protein complexes contain paralogous proteins at their cores; the duplication of a self-binding protein leads to dimerization of the paralogous proteins and subsequent recruitment of additional subunits. Gene duplication after establishment of a heteromeric complex allows subunits to specialize. The plastid caseinolytic protease (Clp) complex provides a model system for studying protein complex evolution, in the context of cytonuclear interactions, gene duplication, and evolutionary rate variation. This complex is highly conserved across bacteria and consists of adaptors, chaperones, and a proteolytic core. It is present in both plastids and mitochondria because these organelles are derived from ancient bacterial endosymbionts. The Clp core contains 14 subunits; in mitochondria and most bacteria, all 14 subunits are encoded by the same gene. However, in the cyanobacterial and plastid lineage, multiple rounds of gene duplication have led to a core encoded by nine different genes in the model plant species Arabidopsis thaliana. Further, only one of these plastid Clp core subunit genes is encoded by the plastid itself—the remaining eight are encoded by the nucleus, the result of gene transfers from the organelle to the nucleus early in the history of green plants. In addition to representing multiple rounds of gene duplication, the plastid Clp core also demonstrates extreme rate variation across green plants. The plastid-encoded subunit (ClpP1) is typically highly conserved across species. However, in some species, ClpP1 is one of the most rapidly evolving genes across all three genomes. In this dissertation, I use these features of the plastid Clp complex to shed light on protein complex evolution in various contexts. After a general introduction to the field in Chapter 1, Chapter 2 focuses on the evolutionary history of ClpP1, looking at rate variation and the loss of introns, RNA editing sites, and catalytic sites across green plants. Through mass spectrometry, I determine that ClpP1 is still a functional protein in Silene noctiflora, which has one of the most divergent plastid Clp complexes known. This work also includes an evolutionary rate covariation analysis between ClpP1 and the nuclear-encoded Clp core genes. Chapter 3 provides genomic resources, including a high-quality, long-read transcriptome, for S. noctiflora, which is a species of interest for the reason outlined above. Analysis of the transcriptome revealed a triplication of one of the nuclear-encoded Clp core genes in this species. Chapter 4 discusses the recent duplication history of the nuclear-encoded Clp core genes across a broad range of flowering plants. I use these data to examine and characterize post-duplication evolutionary fates of paralogs. These analyses are extended to another plastid complex, acetyl-CoA carboxylase (ACCase). Taken together, these chapters elucidate various features of plastid Clp complex evolution as well as provide insight into the possible causes and consequences of rate variation and gene duplication in the coevolution of protein complex subunits.