Browsing by Author "Dayan, Franck E., advisor"
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Item Embargo Involvement of CYP72A219 in herbicide-resistant Palmer amaranth and the role of P450 reductase in the mechanism of metabolic resistance(Colorado State University. Libraries, 2023) Rigon, Carlos A. G., author; Gaines, Todd A., advisor; Dayan, Franck E., advisor; Beffa, Roland, committee member; Peebles, Christie, committee memberHerbicide resistance in weeds poses a major challenge to modern agriculture worldwide, impacting effective weed control strategies. Metabolic resistance stands out as the major and more complex resistance mechanism due to its ability to metabolize a wide range of herbicides within weed species. Metabolic resistance involves herbicide metabolism through three key phases: activation, conjugation, and sequestration. These phases involve the action of important enzymes such as cytochrome P450 monooxygenases, glutathione S-transferases, and ABC transporters. Metabolic resistance mechanisms have gained prominence in the past decade, posing significant challenges to sustainable agriculture and weed management practices. Amaranthus palmeri (Palmer amaranth) one of the most troublesome weeds globally has evolved metabolic resistance to HPPD inhibitor tembotrione. Understanding and addressing the mechanism are crucial for developing effective strategies to combat herbicide resistance and ensure global crop production. In the present study, four upregulated P450 genes were identified in HPPD-resistant Palmer amaranth from Nebraska (NER), a troublesome weed species. Among these genes, CYP72A219_4284 demonstrated the ability to deactivate the herbicide tembotrione in a heterologous system. This gene was also upregulated in metabolic HPPD-resistant Palmer amaranth plants from different fields across the United States, indicating its involvement in conferring herbicide resistance. Our study also investigated the regulation of these resistance genes, including the promoter sequences and transcription factors involved. Additionally, quantitative trait loci associated with herbicide resistance were identified. This work represents the first identification and validation of genes responsible for herbicide metabolism in Palmer amaranth. Validation of the metabolic resistant gene and the exploration of regulatory mechanisms contribute to a better understanding of metabolic herbicide resistance in weeds, facilitating the development of effective weed management strategies. Cytochrome P450 reductase (CPR), an essential enzyme localized in the endoplasmic reticulum, provides electrons for P450 enzymes during monooxygenase reactions. The transfer of electrons from NADPH to the P450 active site occurs through a complex CPR:P450 interaction. Despite the numerous P450 genes in plant genomes, CPR genes are limited, typically consisting of two or three copies. In Arabidopsis, the two CPR genes, ATR1 and ATR2, have distinct roles in primary and inducible metabolism, respectively. Our study investigated the function of ATR1 and ATR2 in transgenic Arabidopsis plants overexpressing the CYP81A12, which is known to metabolize a wide range of herbicides. The hypothesis was that silencing these ATR1 or ATR2 genes would lead to a reduction of P450 activity involved in herbicide metabolism. ATR1 predominantly transfers electrons to CYP81A12, as knocking down ATR1 led to a significant reduction in herbicide resistance. Knockouts of the ATR2 gene also resulted in decreased herbicide resistance, although the effect was less pronounced. Variation in the number and function of CPR genes among different weed species suggests diverse genetic pressures and potential targets for herbicide resistance management. Inhibition of CPR activity could be a promising approach to restore herbicide effectiveness against metabolic herbicide-resistant weeds. This is the first study to our knowledge that explores the involvement of CPR genes in herbicide resistance in weeds, providing valuable insights into their crucial role. The findings significantly advance our understanding of the mechanisms underlying CPR-mediated herbicide resistance and offer potential targets for the development of effective weed management strategies.Item Open Access Physiological and biochemical mechanisms behind the fast action of glufosinate(Colorado State University. Libraries, 2019) Kagueyama Takano, Hudson, author; Dayan, Franck E., advisor; Westra, Philip, advisor; Reddy, Anireddy, committee member; Preston, Christopher, committee member; Gaines, Todd, committee memberGlufosinate is one of the few herbicides that are still effective for controlling herbicide resistant weeds, but its performance is often inconsistent and affected by environmental conditions. It inhibits glutamine synthetase (GS) by competing with glutamate for the active binding site. Unlike other amino acid biosynthesis inhibitors, glufosinate is a fast-acting herbicide and susceptible plants develop visual symptoms within a few hours after treatment. Inhibition of GS leads to ammonia accumulation and photosynthesis inhibition, which have traditionally been proposed as the causes of the rapid phytotoxicity. This dissertation presents a new understanding of the mechanism(s) of action of glufosinate and a biochemical approach to improve its herbicidal efficacy. Glufosinate uptake is inhibited by glutamine levels in the plant, and translocation is not affected by the rapid phytotoxicity. Glufosinate translocates primarily through the apoplast (xylem) rather than the symplast (phloem) probably due to its physicochemical properties and the absence of an effective membrane transporter. Glufosinate efficacy is proportional to the herbicide concentration in leaf tissues. Neither ammonia accumulation nor carbon assimilation inhibition are directly associated with the fast action of glufosinate. Instead, rapid phytotoxicity results from a massive light-dependent accumulation of reactive oxygen species (ROS). Inhibition of GS blocks the photorespiration pathway leading to a massive photooxidation damage. Under full sunlight, the excess of electrons is accepted by molecular oxygen leading to ROS generation. These free radicals cause lipid peroxidation, which ultimately leads to rapid cell death. The addition of protoporphyrinogen oxidase (PPO) inhibitors to glufosinate enhances ROS accumulation and herbicidal activity. This enhanced activity results from protoporphyrin formation at high levels due to a transient accumulation of glutamate, the precursor for chlorophyll biosynthesis. The herbicide combination also showed enhanced activity in the field and may help to overcome the lack of glufosinate efficacy under certain environmental conditions.Item Open Access Quizalofop-resistant wheat: biochemical characterization of the AXigen™ trait and corresponding metabolism(Colorado State University. Libraries, 2021) Bough, Raven A., author; Dayan, Franck E., advisor; Gaines, Todd A., committee member; Haley, Scott, committee member; Pearce, Stephen, committee memberA new weed management tool in wheat, the CoAXium™ Wheat Production System, incorporates quizalofop-resistant wheat, a specialized formulation of quizalofop (Aggressor™), and a stewardship management program for effective management of annual grasses with otherwise limited control options. The AXigen™ trait confers resistance primarily through a single-point mutation in ACC1. The mutation causes an alanine to valine substitution at position 2004 in wheat acetyl-CoA carboxylase (ACCase) relative to the Alopecurus myosuriodes reference. Through greenhouse and biochemical studies paired with protein homology modelling and simulations, the research presented herein provides strong evidence that a conformational change imparted by the amino acid substitution results in quizalofop-resistant ACCase. Conversely, the mutation conveys negative cross-resistance to haloxyfop, a similar herbicide to quizalofop with a smaller molecular volume. The remaining research objectives focus on quizalofop metabolism in CoAXium™ wheat. Liquid chromatography-mass spectrometry measurements of quizalofop content over time from liquid demonstrate cooler temperature conditions (4.5°C) delay quizalofop metabolism by 4 times compared to warmer temperature conditions (19°C). Reduced temperatures also delay quizalofop metabolism to the same extent in the following annual grass weed species: Aegilops cylindrica, Bromus tectorum, and Secale cereale. Further, additional studies suggest herbicide metabolism mechanisms enhance overall CoAXium™ wheat quizalofop resistance. Despite similar ACCase resistance, resistant winter and spring wheat varieties convey varying degrees of whole-plant resistance. In winter wheat but not spring wheat, increased resistance corresponds to a shorter quizalofop half-life, implying faster metabolism boosts overall resistance. Treatment of resistant spring wheat varieties with cloquintocet, a metabolism-boosting safener, increases overall resistance. Follow-up differential expression analysis of cloquintocet-treated plants may support differential metabolism findings and lead to identification of putative candidate genes associated with upregulated herbicide metabolism, such as cytochrome P450 monooxygenases, glutathione-S-transferases, and glycosyltransferases.