Browsing by Author "Dayan, Franck E., advisor"

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Open Access
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 member
Herbicide 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.
Open Access
Molecular mechanisms of herbicide resistance in rice and kochia
(Colorado State University. Libraries, 2024) Gupta, Srishti, author; Dayan, Franck E., advisor; Gaines, Todd A., advisor; Reddy, Anireddy, committee member; Kumar, Vipan, committee member
Herbicide stress is an important challenge in agriculture and understanding how plants respond to herbicide exposure is crucial for developing effective weed management strategies. Transcription factors (TFs) play a pivotal role in regulating gene expression and mediating plant responses to various environmental stimuli, including herbicide stress. This dissertation aimed to elucidate the role of TFs in herbicide tolerance and sensitivity across plant species. A brief introduction was provided in Chapter 1. Subsequently, by analyzing transcriptomic data from different studies, we identified key TFs involved in herbicide responses. Our findings in Chapter 2 revealed distinct TF signatures, including bZIP, NAC, WRKY, and ERF, that were consistently upregulated in herbicide-tolerant plants. associated with herbicide tolerance or sensitivity, suggesting potential regulatory mechanisms in metabolic pathways and downstream signaling. These results underscore the importance of complex interplay between herbicide class, treatment duration, and plant species on TF expression patterns. In Chapter 3, we focused on herbicide resistance in rice, a critical staple crop. Transcriptomic analysis revealed upregulation of key detoxification genes, including glutathione S-transferase (GST) and cytochrome P450 (CYP450), in the NTSR mutant, suggesting their involvement in herbicide metabolism. Functional characterization confirmed increased glutathione S-transferase activity in the NTSR genotype. Additionally, computational studies identified a novel transcription factor, ZOS-1-16, with a potential role in regulating herbicide response. We investigated a novel non-target site resistance (NTSR) mechanism conferred by a mutation in the transcription factor ZOS-1-16. Our findings demonstrated that ZOS-1-16 upregulates genes like GSTs and CYPs involved in herbicide detoxification, leading to increased resistance to the herbicide quizalofop-p-ethyl (QPE). This study highlights the potential of targeting TFs for developing herbicide-resistant rice varieties. Finally, Chapter 4 explored glyphosate resistance in the invasive species Bassia scoparia (kochia). We investigated the inheritance of glyphosate resistance in kochia populations and found that it is primarily due to an increase in the copy number of the EPSPS (5‐enolpyruvyl‐3‐shikimate phosphate synthase) gene. Additionally, we estimated the outcrossing rate of kochia under field conditions and found a high level of outcrossing, which contributes to the rapid spread of glyphosate-resistant biotypes. Overall, this dissertation provides valuable insights into the role of TFs in herbicide responses and highlights the potential for developing novel strategies to enhance herbicide tolerance and manage herbicide-resistant weeds.
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 member
Glufosinate 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.
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 member
A 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.
Open Access
Unravelling the resistance mechanism to dicamba in Palmer amaranth (Amaranthus palmeri)
(Colorado State University. Libraries, 2024) Moreno Serrano, Dustin Abdiel, author; Dayan, Franck E., advisor; Gaines, Todd A., committee member; Schipanski, Meagan, committee member
Auxin-mimic herbicides (AMHs) have been widely used for more than 70 years, primarily for control of pernicious broadleaf weeds and a few grassy weeds. So far 87 weeds have evolved resistance to AMHs, and it is expected to continue to increase over time. Resistance mechanisms to AMHs are not well understood and most of the reported cases have not been investigated to determine each individual resistance mechanism. Herbicide resistance mechanisms are classified into two main branches. Target-site-resistance (TSR) is when a mutation in herbicide binding site prevents the herbicide from interacting with the enzyme or when overexpression of the herbicide target site results in more enzyme than what the herbicide can inhibit in the plant cell. Non-target-resistance (NTSR) include enhanced herbicide metabolism, reduced absorption, altered translocation, and sequestration. In this current research, resistance to dicamba in a population of Amaranthus palmeri (Palmer amaranth) was investigated. In 2020 a population of the troublesome weed Amaranthus palmeri (Palmer amaranth) from Lauderdale county in Tennessee, USA, with a 12-fold resistance to dicamba was identified. Metabolism of dicamba was evaluated and tested using inhibitors of important enzymes involved in herbicide detoxification (e.g., cytochrome P450 monooxygenase and glutathione S-transferases). There was no difference in dicamba metabolism between the resistant Lauderdale (R_PA) and susceptible Arizona (S_PA) populations. RNA-seq study was conducted to investigate potential mutations in AUX/IAAs, which are transcriptional repressors. They regulate transcription factors like Auxin Response Factors (ARFs), and are also co-receptors of auxin-mimic herbicides, and are involved in the regulation of auxin response genes. A mutation in co-receptors can lead to auxin-mimic herbicide resistance; however, there were no mutations in 18 AUX/IAAs and also in other important proteins such as Transport Inhibitor Response 1 (TIR1), Auxin Binding Protein (APB), and Auxin Signaling F-box (AFB). In addition, auxin-response genes responded similarly or differently to dicamba in treated biotypes. Nevertheless, it is noteworthy that the expression of some AUX/IAAs genes changed after dicamba treatment in sensitive plants but not in resistant plants, especially AUX/IAA29. The results suggest that a physiological response is not primarily involved in the resistance mechanism to dicamba because no significant differences in dicamba metabolism were identified, suggesting that dicamba is broken down to less active metabolite, but at the same rate in both R_PA and S_PA. Additionally, no epinasty was observed in resistant plants, a common response when TSR is involved as a primary mechanism. PIF3/4, which is a key transcription factor involved in regulating plant development and response to light, responded to dicamba treatment in sensitive plants, while not responding in resistant plants after dicamba application. Also, expression of AUX/IAA29 did not respond in resistant plants, which is directly involved in the activation of PIF3/4, a transcription factor involved in auxin perception. We hypothesize that PIF3/4 may be involved in the resistance mechanism to dicamba through auxin signaling and/or regulation. However, this hypothesis should be validated by molecular techniques to confirm it. This dicamba-resistant Palmer amaranth biotype has a novel resistance mechanism that remains to be fully elucidated.