Browsing by Author "Gaines, Todd A., advisor"

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Open Access
Aspects of weed resistance to auxinic herbicides
(Colorado State University. Libraries, 2020) Rodrigues Alves de Figueiredo, Marcelo, author; Gaines, Todd A., advisor; Argueso, Cristiana T., committee member; Dayan, Franck E., committee member; Reddy, Anireddy S. N., committee member
Synthetic auxins have been widely used for selective control of broadleaf weeds since the mid-1940s. After more than 70 years using synthetic auxin herbicides, there are 41 different resistant species reported. Weed resistance to auxin herbicides is poorly understood and in most reported cases, no studies have been done to investigate the mechanistic changes that occur in resistant populations. The mechanisms of herbicide resistance in weeds are classified as 1) target-site when mutations reduce the interaction of the herbicide molecule to its binding site and/or changes of gene expression of the targeted enzyme to compensate for herbicide inhibition; and as 2) non-target-site mechanisms, which include any genetic mutations that will prevent or reduce the herbicide reaching its site of action. In this present research, two 2,4-D resistant weed species were studied, and the mechanisms of resistance were elucidated, where one species evolved metabolic non-target-site resistance to 2,4-D and the second species evolved a novel mechanism of target side modification. In 2009, an Amaranthus tuberculatus (common waterhemp) population with ten-fold resistance to 2,4-D was found in Nebraska, USA. Using the same 2,4-D-resistant and a known susceptible A. tuberculatus population from Indiana, the mechanism of 2,4-D resistance was examined by conducting [14C] 2,4-D absorption, translocation and metabolism experiments. No differences were found in 2,4-D absorption, but resistant plants translocated more of the radioactive material than susceptible A. tuberculatus. Resistant plants metabolized [14C] 2,4-D more rapidly than susceptible plants. The main metabolites were purified and their structures were solved by NMR and HRMS. Susceptible plants conjugate 2,4-D to 2,4-D Aspartic Acid (2,4-D-Asp). Resistant plants showed a distinct metabolic profile where 2,4-D is hydroxylated into 5-OH-2,4-D, conjugated in a sugar metabolite (5-OH-2,4-D-Glucoside) and malonylated into 5-OH-2,4-D-(6-O-Malonyl)-Glucoside. Pre-treatment with the cytochrome P450 inhibitor malathion inhibited 2,4-D hydroxylation. Toxicological studies in waterhemp and Arabidopsis confirmed that the hydroxylated metabolite lost its auxinic action and toxicity. In contrast, the 2,4-D-Asp metabolite induced auxin inhibition to the plants tested. These results demonstrate that resistant A. tuberculatus evolved novel detoxification reactions that rapidly metabolize 2,4-D, potentially mediated by cytochrome P450. That novel mechanism is more efficient and produces metabolites with lower toxicity compared to the innate aspartic acid conjugation. Metabolism-based herbicide resistance poses a serious challenge for weed management due to the potential for cross-resistance to other herbicides. Sisymbrium orientale (Indian hedge mustard) is an important weed species in Australia reducing yields in crops and pastures. In 2005, a 2,4-D and MCPA resistant population was reported in the Port Broughton region in South Australia. Aux/IAAs are dynamic repressor proteins that regulate Auxin Response Factors (ARFs) to activate auxin related genes and are also co-receptors for auxins and synthetic auxin herbicides. The degradation of Aux/IAAs is done by the enzyme complex E3, called SCFTIR1/AFB, which, in the presence of auxin, performs ubiquitination on Aux/IAA making it a target of proteasome 26S, an enzyme responsible for proteolysis in eukaryotes. An RNAseq study showed that a 27 bp deletion in Aux/IAA2 (IAA2) degron tail was correlated to the resistant phenotype. The mutant allele was functionally validated to confer 2,4-D resistance by transforming Arabidopsis thaliana with the wild type SoIAA2 and SoIAA2Δ27 alleles. Performing binding analysis by surface plasmon resonance, the association of TIR1 in the presence of auxin (IAA, 2,4-D and dicamba) showed slower association and faster dissociation to the resistant IAA2 peptide compared to the susceptible IAA2 peptide. Our results suggest that the loss of 9 amino acids located in the degron tail may reduce the capacity of IAA2 to "embrace" TIR1 in the presence of auxin, reducing ubiquitination rate, resulting in higher stability to repress auxin response factors and ultimately conferring resistance to 2,4-D.
Open Access
Decreased dicamba transport due to increased flavonoid biosynthesis: a candidate dicamba resistance mechanism
(Colorado State University. Libraries, 2016) Pettinga, Dean J., author; Gaines, Todd A., advisor; Ward, Sarah, committee member; Sloan, Daniel, committee member
Resistance to dicamba (a synthetic auxin herbicide) has been documented in Kochia scoparia (L.) Schrad. populations since 1994, but the molecular mechanisms of observed resistance cases remain elusive. An RNA-Seq approach was used to identify transcripts with significantly differential transcription responses between inbred lines of dicamba-resistant (9425R) and dicamba-susceptible (7710S) K. scoparia in response to dicamba application. Among the significantly differentially expressed transcripts was both Chalcone Synthase (CHS), the first enzyme and rate-limiting step in the flavonoid biosynthesis pathway, and Flavono 3'-Hydroxylase (F3'H), which catalyzes the conversion of quercetin into kaempferol, known inhibitors of auxin transport. In silico expression patterns of both transcripts were confirmed with qRT-PCR. An F2 population derived from a cross of 9425R x 7710S segregating for the resistance phenotype was assayed for CHS and F3'H expression using qRT-PCR. Dicamba-resistant F2 individuals displayed significantly higher CHS transcript abundance compared to dicamba-susceptible F2 individuals, associating the resistance phenotype of 9425R with a greater overall flux through the flavonoid biosynthesis pathway. Increased production of the auxin transport inhibitors quercetin and kaempferol could reduce intercellular transport and vascular loading of dicamba, causing a substantial reduction in dicamba efficacy by reducing its translocation to sensitive meristematic tissue, thereby conferring the observed resistance phenotype.
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 genetics of herbicide resistance in Palmer amaranth (Amaranthus palmeri): metabolic tembotrione resistance and geographic origin of glyphosate resistance
(Colorado State University. Libraries, 2018) Küpper, Anita, author; Gaines, Todd A., advisor; Dayan, Franck E., committee member; Nissen, Scott J., committee member; Reddy, Anireddy S. N., committee member
Palmer amaranth (Amaranthus palmeri) is a major weed in U.S. cotton and soybean production systems, partly because it evolved resistance to five different herbicide modes of action. Resistance to the 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibitor tembotrione in a population from Nebraska (NER) is due to enhanced metabolism. This type of non-target-site resistance is especially troublesome because of its potential for cross-resistance. Tembotrione-susceptible (NES) and NER formed the same tembotrione metabolites but NER exhibited faster 4-hydroxylation followed by glycosylation. The T 50 value (time for 50% production of the maximum 4-hydroxylation product) was 4.9 and 11.9 h for NER and NES, respectively. Hydroxylation is typically catalyzed by cytochrome P450 monooxygenases (CYPs). Metabolism differences between NER and NES were most prominent under 28°C conditions and herbicide application at the four-leaf stage. An RNA-Seq transcriptome analysis was conducted with Pseudo-F 2 tembotrione-resistant and -susceptible individuals originating from three separate NER x NES crosses that were sampled before, six, and twelve h after treatment (HAT). Differential gene expression analysis identified CYP72A219 and CYP81E8 as strong candidates for metabolic resistance. The contigs were constitutively expressed in resistant plants, as were the contigs for several glycosyltransferases (GTs), oxidase, and glutathione-S-transferase (GST). Exposure to tembotrione further increased their expression in both resistant and susceptible plants. Originally native to the Southwest, A. palmeri has spread throughout the country. In 2004 a population was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant crop systems. Glyphosate resistance in the species is now highly prevalent in USA and was also discovered in Brazil in 2015. This was confirmed by species identification with a genetic marker, dose-response studies, shikimate accumulation assay, and EPSPS copy number assay. The Brazilian population was also resistant to sulfonylurea and imidazolinone ALS inhibitor herbicides conferred by two different alleles for target-site mutations in the ALS gene (W574L and S653N). The degree of genetic relatedness among eight different populations of glyphosate-resistant (GR) and –susceptible (GS) A. palmeri from various geographic regions in USA was investigated by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimate accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.
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
Understanding weed biology and herbicide resistance to improve weed management
(Colorado State University. Libraries, 2020) Soni-Castillo, Neeta, author; Gaines, Todd A., advisor; Dayan, Franck E., committee member; Argueso, Cristiana T., committee member; Haley, Scott D., committee member
Weed management is essential in agriculture, natural areas, and rangelands. Weed control has mainly relied on herbicides. These chemical compounds are a low-cost option, easy to apply, and very efficient to eliminate weeds. However, as part of survival strategies weed species have evolved mechanisms to overcome herbicides and continue their life cycle. Thus, it is imperative that we increase our knowledge in weed biology and resistance mechanisms to develop better management strategies. Here I present three chapters that cover these areas of study. First, as an intent to promote more tools for management strategies in winter wheat, a field survey was conducted to identify the potential to implement harvest weed seed control for problematic winter annual grasses in this cropping system. The second chapter covers the results of a herbicide resistance survey to screen for imazamox and quizalofop resistance of troublesome winter annual grasses in winter wheat and rangeland areas. The third chapter aimed to determine the distribution of native and introduced Phragmites australis haplotypes which is a riparian species problematic in rangeland and natural areas. Harvest weed seed control methods showed potential to manage downy brome, feral rye, and jointed goatgrass. Seed retention of these winter annual grasses was over 75% indicating that the majority of seeds could be collected during wheat harvest. After screening over 280 samples of winter annual grasses, only two feral rye populations showed resistance to imazamox. Further studies on resistance mechanisms showed that one population (A) can rapidly metabolize the herbicide compared to a susceptible and the second population (B) contained a target site mutation in the imazamox target enzyme. Introduced Phragmites australis haplotypes were identified in Colorado using molecular markers. In addition, a low-cost and quick genotyping tool was developed to encourage land managers to conduct more frequent monitoring. Main results from this dissertation are expected to contribute with the big endeavor of promoting integrated weed management solutions and better weed biology understanding.