Browsing by Author "Dayan, Franck, advisor"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
Item Open Access Factors contributing to herbicide response in CoAXium wheat(Colorado State University. Libraries, 2024) Pelon, Amber L., author; Dayan, Franck, advisor; Gaines, Todd, committee member; Schipanski, Meagan, committee memberCompared to other pests, weed competition has the most significant negative impact on wheat grain yield. Understanding the contribution of metabolism in overall tolerance to herbicides can lead to new methods for controlling weeds in wheat. Glutathione S-transferase's (GSTs) role in the detoxification of herbicides has been studied since 1970. Previous literature reported increased resistance to herbicides with higher GST activity in black grass (Alopecurus myosuroides) and Asia minor bluegrass (Polypogon fugax). Resistance could be reversed by inhibiting GST activity. This research assesses the role of Phase 2 plant cell metabolism by testing (GST) inhibition to see if it influences the metabolism of quizalofop P-ethyl (QPE) in winter wheat (Triticum aestivum). We hypothesized that the addition of a safener would make the wheat more tolerant to the herbicide while the addition of a GST inhibitor would make the wheat more sensitive to QPE. Experiments were conducted analyzing the QPE effect on whole-plant biomass and an LC-MS/MS analysis of the amount of quizalofop acid (QZA) found in plant extracts. Safeners enhanced herbicide metabolism which increased CoAXium wheat tolerance to QPE. GST inhibitors, conversely, decreased herbicide metabolism causing CoAXium wheat to be more sensitive to QPE. Understanding the contribution of metabolism in overall resistance to herbicides can lead to breeding improvements for more herbicide-tolerant wheat varieties and new methods for controlling weeds in wheat.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 Embargo The need for new inhibitors of photosynthesis in agricultural settings, and the novel herbicidal compound AS9057(Colorado State University. Libraries, 2024) Twitty, Alyssa, author; Dayan, Franck, advisor; Abdel-Ghany, Salah, committee member; Phillip, Yael, committee memberDue to increased food demand, the need for use of herbicides is both necessary and on the rise. Several herbicide classes target photosynthetic electron transport: HRAC Groups 5, 6, and 22. These herbicides are used in large amounts in many different cropping systems to control several species of broadleaf and grass weeds. The first chapter provides a comprehensive review of what these photosynthesis inhibitors are, how they are used and their mode of action. Presently, commercial herbicides only inhibit electron flow at two different sites (PSII and PSI). Those which inhibit electron flow at PSII block the movement of electrons down the electron transport chain, while those which inhibit at PSI accept electrons. Necrosis developing on the leaves of plants treated with PSII and PSI inhibitors is due to the accumulation of reactive oxygen species. Evolution of resistance, toxicity concerns, and other limitations of these herbicides call for the exploration of new chemistries that can be used to target this pathway. One of these new chemistries has been identified as AS9057. AS9057 is a natural product identified as a novel herbicide with a potentially new mode of action using AI4AI, an AI platform for herbicide discovery developed by Agrematch. Greenhouse trials demonstrated that the herbicidal activity of AS9057 was light-dependent. The rapid burndown symptoms-developing on treated plants, combined with its chemical structure, suggested that AS9057 may target photosystem II. Measurements of photosynthetic electron transport rates in treated plants alongside data from oxygen evolution assays did not support this hypothesis. Further experiments suggested the AS9057 may instead act as an electron diverter. Oxygen consumption assays in isolated thylakoid membranes using a variety of electron transport inhibitors revealed that AS9057 likely acts on photosystem I in a similar manner to paraquat, but at a potentially new step between P700 and NADP+. This is consistent with other reports that AS9057 can act as an electron acceptor for flavoproteins. Ferredoxin-NAPD+ reductase is a flavoprotein with a redox potential similar to that of AS9057. Thus, it is currently hypothesized that AS9057 acts as an electron acceptor at or near the ferredoxin to form a radical and generate reactive oxygen species which causes the light-dependent herbicidal effect which is observed in treated plants from greenhouse trials.Item Open Access Xenobiotics translocate in aquatic plants: a case study using three aquatic herbicides(Colorado State University. Libraries, 2022) Ortiz, Mirella F., author; Dayan, Franck, advisor; Nissen, Scott, advisor; Bedinger, Patricia, committee member; Heilman, Mark, committee memberWhen invasive aquatic weeds dominate aquatic ecosystems there are numerous negative impacts. Milfoil (Myriophyllum spp.) and hydrilla [Hydrilla verticillata (L.f.) Royle] are the most costly aquatic plants to manage in the U.S. per year. These invasive plants form extensive surface canopies that negatively affect water quality and native plant communities, and can also impact recreational uses such as swimming, fishing, and boating. Synthetic auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D), have been widely used for selective control of milfoil since 1959. Since then, several populations of hybrid watermilfoil (M. sibiricum x M. spicatum; HWM) have showed lower sensitivity to this herbicide. In 2015, a HWM population with lower sensitivity to 2,4-D was found in Idaho, USA. Using the same 2,4-D-resistant population and a known susceptible Eurasian watermilfoil (M. spicatum; EWM) population from Colorado, the mechanism of 2,4-D resistance was examined by conducting 14C-2,4-D absorption, translocation, desorption, and metabolism experiments. 2,4-D resistance in HWM is not due to non-target-site resistance as no differences in herbicide absorption, translocation, desorption and/or metabolism were identified; therefore, target-site resistance is the most likely resistance mechanism. More research is needed to identify the molecular basis for the 2,4-D-resistant trait in HWM. Herbicide combinations are widely recommended to alleviate the evolution of herbicide resistance. The aquatic herbicide endothall is often used in combination with 2,4-D for HWM management as an effective control option and a resistance management strategy, but it is still unknown how combining herbicides might impact the behavior of each herbicide. Experiments combining radiolabeled with non-radiolabeled herbicides were conducted to evaluate herbicide absorption, accumulation, and translocation from shoots to roots in HWM. Endothall accumulation was not impacted when these herbicides were applied in combination, but its translocation from shoots to roots was reduced by 50% when applied in combination with 2,4-D. When 2,4-D, was applied in combination with endothall shoot absorption increased by 80%; however, 2,4-D movement from shoots to roots was reduced from 24.8% ± 2.6 to only 3.93% ± 0.4 when in the presence of endothall. The overreliance on a single mode of action resulted in evolved fluridone resistance in hydrilla in the late 1990s. 2,4-D is not effective for hydrilla control at label rates, but the most recently registered auxinic herbicide, florpyrauxifen-benzyl, is highly active against hydrilla. Where fluridone-resistant hydrilla is present, endothall is being used in combination with florpyrauxifen-benzyl for its control. In order to test experiments combining radiolabeled and non-radiolabeled endothall and florpyrauxifen-benzyl were conducted to evaluate herbicide absorption, accumulation, and translocation in two hydrilla biotypes, monoecious (MHV) and dioecious (DHV). Herbicide accumulation in both biotypes was not impacted when these herbicides were applied in combination. Endothall translocation from shoots to roots in DHV did not appear to be impacted (alone = 18.7% ± 1.4; combination = 23.2% ± 2.2); however, endothall shoot-to-root translocation in MHV was reduced from 16.2% ± 1.3 to 2.2% ± 0.1 when applied in combination with florpyrauxifen-benzyl. Florpyrauxifen-benzyl shoot-to-root translocation was reduced by 16 and 6 times in DHV and MHV when applied in combination with endothall, respectively. These data highlight differences in herbicide behavior when herbicides are applied in combination. Future research is needed to determine if these differences negatively impact the operational effectiveness when herbicides are applied in combination. Lastly, endothall and 2,4-D have been used to control aquatic weeds for more than 60 years, and still there is very little information available about the in planta behavior of these herbicides in aquatic weed species. 2,4-D is purportedly systemic in aquatic plants based almost entirely on its behavior in terrestrial plants. It was demonstrated in this dissertation that radioactive 2,4-D and endothall can translocate from shoots to root systems; however, it was not determined if the radioactivity in the roots was parent herbicide or a metabolite(s). Therefore, the last chapter of this dissertation used multiple analytical methods to answer the question if 2,4-D and endothall are truly systemic in aquatic plants. The intact 2,4-D detected in HWM shoots was 1.31 µg g-1 dry weight (DW) and 0.11 µg g-1 DW was detected in the roots. For endothall, 1.08 and 0.12 µg g-1 DW was detected in DHV shoots and roots, respectively. We therefore conclude that 2,4-D and endothall have similar in planta behavior, with about 8-10% of absorbed intact active ingredient translocating to the roots of these aquatic plants.