Progress 01/01/24 to 12/31/24
Outputs
Target Audience:The project addresses a critical problem in the row crop production systems of the Southern US, such as soybean, corn, and cotton. Glyphosate-resistant Amaranth (Amaranthus palmeri) is one of the most aggressive and problematic weed species in modern agriculture, resulting in >70% yield loss if left unmanaged and resulting in billions of dollars in economic loss. Growers incur an additional $50-$75 per acre to manage herbicide-resistant amaranth. Thus, glyphosate-resistant Palmer amaranth has led to substantial economic burdens for farmers, with increased management costs and significant yield losses, underscoring the need for integrated weed management approaches. Thus, the project serves the needs ofgrowers, academia and industry alike. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student was trained in the various aspects of the project, including enzyme assays, metabolomic experiments, data processing, and manuscript preparation. Three undergraduates were trained in treatment application, sample collection and processing. Eight graduate students were trained on the planning and execution and metabolomic experiments. How have the results been disseminated to communities of interest?The results of the project were presented at regional and national conferences, in classrooms via formal lectures, and to the grower communities via field visits and one-on-one interactions. What do you plan to do during the next reporting period to accomplish the goals?During the next year of the project, we plan to complete the metabolomics-based dose-response studies and also to complete the testing of additional enzyme inhibitors to dampen the phytochemical-mediated stress-resilience of this weed.
Impacts
What was accomplished under these goals?
Metabolic flux analysis to identify the physiology related to herbicide phytotoxicity Despite known primary effects, secondary metabolic consequences remain poorly understood, particularly how glyphosate influences both aromatic and non-aromatic amino acid metabolism. We utilized stable isotope-resolved metabolomics (SIRM) to differentiate the contributions of anabolic (de novo synthesis) and catabolic (protein degradation) pathways to amino acid pools in glyphosate-resistant (R) and glyphosate-susceptible (S) Amaranthus palmeri biotypes. Plants were grown in the greenhouse and provided with isotope-labeled nitrogen after a sublethal dose of herbicide application. Key results: Significant elevation (approximately two-fold) of total amino acid pools in susceptible plants post-glyphosate application; negligible changes in resistant plants. Increased amino acid concentrations in susceptible plants primarily due to enhanced protein degradation, evidenced by substantial reduction in total soluble proteins. Elevated abundance of both existing (14N) and newly synthesized (15N) amino acids in susceptible biotypes, indicating concurrent increases in catabolic processes and de novo synthesis. Marked decrease in aromatic amino acids in glyphosate-treated susceptible biotypes, aligning with targeted shikimate pathway inhibition. Major increases observed in shikimate-independent amino acids, particularly asparagine, glutamine, alanine, and serine, suggesting significant contributions from de novo synthesis. Susceptible biotypes exhibited a substantial increase in glutamine/glutamate (Gln/Glu) ratio, indicative of disrupted nitrogen assimilation and decreased glutamate synthesis due to limited α-ketoglutarate availability. Resistant biotypes demonstrated minimal metabolic disruption, maintaining stable amino acid pools, protein content, and nitrogen assimilation ratios, reflecting robust metabolic resilience. This research offers critical insights into broader metabolic impacts of glyphosate, clearly distinguishing differential metabolic adaptations in resistant versus susceptible weed biotypes. By employing advanced metabolomics, the study provides valuable knowledge essential for managing herbicide resistance in agricultural practices. Metabolomics as a tool to understand stress priming and modes of action of herbicide. Herbicide resistance is an increasing challenge in agriculture, significantly contributing to global crop yield losses. A detailed understanding of herbicide modes of action (MoA) is essential for effective resistance management. Dose-response metabolomics represents a powerful methodology for MoA identification, yet managing large, complex datasets remains a significant barrier. In this study, we investigated the TOXcms approach--an independent software tool integrating dose-dependent metabolomics data with physiological parameters such as biomass, respiration, and photosynthesis--in the context of weed science to identify both primary and secondary herbicide effects. To assess the efficacy of TOXcms, we applied this approach to two contrasting biotypes of Amaranthus palmeri: glyphosate-resistant (CoR) and glyphosate-susceptible (CoS). Each biotype was treated with 12 doses of glyphosate ranging from 0 to 6.4 kg acid equivalent (a.e.) per hectare, with 4 to 6 replicates per dose. Metabolites were profiled using UHPLC/HRMS and GC/MS to capture primary and secondary metabolic compounds. XCMS data processing of LC-MS outputs generated over 10,000 metabolic features per biotype. TOXcms analysis identified 277 dose-responsive features in CoS, of which 223 increased and 54 decreased with escalating glyphosate doses. Importantly, CoS exhibited pronounced metabolic disturbances, whereas CoR presented a comparatively milder response, with only 114 features showing dose-dependent patterns (72 increased, 39 decreased). Putative metabolite identification demonstrated significant disruption in critical primary metabolic pathways within the CoS biotype. Specifically, shikimic acid--a known glyphosate biomarker--showed substantial dose-dependent accumulation (ED?? = 0.31 kg a.e./ha), accompanied by decreased levels of essential amino acids such as L-phenylalanine. Conversely, the CoR biotype exhibited no significant perturbation in shikimate or amino acid metabolism. Instead, stress-related metabolites, including asparagine, monolinolein, acetyl-lysine, and mannitol, were upregulated, suggesting the activation of alternative herbicide tolerance mechanisms. Mummichog pathway enrichment analysis further supported these findings, indicating amino acid metabolism pathway enrichment in CoS. In contrast, CoR demonstrated enrichment in pathways involved in secondary metabolite biosynthesis, arginine-proline metabolism, and nitrogen metabolism. Additionally, CANOPUS classification of TOXcms-identified metabolic features revealed substantial dysregulation within the superclass of "organic acids and derivatives," particularly amino acids and peptide analogues. Although both biotypes exhibited similarities in metabolite classes, CoR demonstrated approximately fourfold fewer dysregulated metabolic features, highlighting its relatively resilient metabolic profile. Overall, this study demonstrates that TOXcms, when integrated with dose-response metabolomics and physiological metrics, provides a robust framework for elucidating herbicide modes of action. Its application in weed science holds potential for gaining novel insights into herbicide-induced metabolic disruptions and developing more effective strategies for managing herbicide resistance. ?Field collection of amaranth seeds: We continue to collect and test the seeds of Palmer amaranth from fields for glyphosate resistance. During the reporting period, seeds from 53 populations of amaranth were collected and screened for the level of glyphosate resistance. To achieve this, seedlings from this population grown in a greenhouse were subjected to the glyphosate treatment application as outlined in the above study. The performance of the seedlings was scored at various time intervals after the treatment. This screening is currently ongoing, and more than 70% of the tested population were found to be resistant to glyphosate at field recommended dosage. Screening chemicals that inhibit secondary metabolite production. We continue to study the feedback inhibition of critical enzymes in Amaranthus players to identify chemicals that could manage the development of resistance. Based on these studies, the chemicals that provide feedback inhibition of the enzymes belonging to the phenylpropanoid pathways predispose the plant to environmental stresses.
Publications
- Type:
Other Journal Articles
Status:
Other
Year Published:
2024
Citation:
Kaur G, Kumar R, Leonard E, Tharayil N. Metabolomics as a complementary conduit to elucidate the mechanisms of priming-mediated stress-memory in plants.(in review)
|
Progress 01/01/23 to 12/31/23
Outputs
Target Audience:The research project is targeted towards addressing the needs of growers in the Southern United States who struggle to manage the surge of herbicide resistance weeds in row crop production systems. The rise in herbicide-resistant weeds is becoming a major challenge for agricultural production worldwide. In the United States, Palmer amaranth [Amaranthus palmeri (S.) Wats.] has merged as the most widespread agronomic weed, in part due to its highly competitive nature and also due to its ability to thwart the best weed control measures, including herbicides. Palmer amaranth is a dioecious annual species native to the southwestern United States and northwestern Mexico. However, in recent years,ithas expanded as far north as Ontario, Canada and as far east as Massachusetts, United States. In 2004, the first case of glyphosate-resistant (GR-) population of Palmer amaranth in the United States was identified in Georgia, and since then, GR-Palmer amaranth populations have been found spread across 28 states. Palmer amaranth causes yield losses ranging from 50-90% in row crop production systems (corn, soybean, and cotton) of the southeastern United States The primary glyphosate resistance mechanism in Palmer amaranth has been identified as the amplification of the gene encoding the EPSPS. Other mechanisms, such as vacuolar sequestration, altered translocation, and target site mutations, have also been reported in Palmer amaranth. On the other hand, whole plant physiology and metabolomics studies have consistently shown glyphosate-induced perturbation of physiology across resistant biotypes, which are not directly connected to perturbation of the shikimate pathway. These physiological perturbations point to the prevalence of off-target toxicity of herbicide and/or a potential time lag in functionalizing the resistance mechanism after the herbicide exposure. The project, by monitoring the physiology of glyphosate in amaranth, aims to elucidate the stress resilience and the potential for resistant biotypes to withstand lethal doses of herbicide through priming and potential management aspects to overcome stress resilence. The results from the study have been presented at three national conferences and incorporated into an undergraduate course on weedy and invasive species. Changes/Problems:The project suffered an 8-month delay in implementation during 2022-2023 due to a weather-related flooding event resulting in lab shutdown. However, the project executions were accelerated after the lab was back functional came online, which helped to overcome the disruption partly. We plan to request a no-cost extension of the project to complete the ongoing experiments. What opportunities for training and professional development has the project provided?One postdoctoral fellow, one research associate, two graduate students and 4 undergraduate students were trained on various research aspects of this project. These include training on plant care, treatment application and associated observation, sample harvest and processing, instrument analysis and data processing. The key outcome from the priming experiments (from the previous reporting period) was incorporated into the undergraduate curriculum on weedy and invasive species (PES 4090/6090). The class discussed the various strategies through which weedy and invasive species circumvent the environmental stress that are otherwise lethal. How have the results been disseminated to communities of interest?The results from this study were presented at three national meetings and one journal article that is currently under internal review. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to continue experiments to identify compounds that modify the phenyl propanoic pathway, which would reduce the stress priming in weeds.
Impacts
What was accomplished under these goals?
EPSPS copy number variation analysis in Palmer amaranth population The primary glyphosate resistance mechanism in Palmer amaranth has been identified as the amplification of the gene encoding the EPSPS enzyme that increases EPSPS transcription and protein activity. To assess the extent of copy number variation (CNVs) of EPSPS, we selected 30 different Amaranth populations for the comprehensive copy number analysis. These biotypes were grown in the greenhouse and half of the plants of each biotype were sprayed with glyphosate. Out of these 30 biotypes, seven biotypes were susceptible, and 23 biotypes were resistant type based on their response to glyphosate. Further, we isolated the DNA from the leaf tissue of 310 glyphosate-treated (GLY) and untreated control plants (CTRL) from 30 biotypes and determined the CNVs using quantitative PCR. An ample amount of variation was observed in these biotypes, ranging from a minimum of one copy in susceptible biotypes to a maximum of 85 copies in the resistant biotypes. Consistent with their phenotype, susceptible plants, on average, have a single copy (0.72), whereas resistant plants have 22 copies (21.57). Resistant biotypes showed a large variation in copy number, varying from >1 to < 85 copies. However, no significant differences were observed in copy number in resistant CTRL and GLY plants, indicating either no or less amplification of EPSPS after the glyphosate treatment. Metabolomic analysis to elucidate the physiological effect of copy number variation Three hundred ten amaranth plants with diverse copy numbers were selected for subsequent untargeted metabolomic analysis. For these plants, 322 primary and 6354 secondary mass features were detected overall (identification with exact mass, retention time, and fragmentation spectrum matching). Principal component analysis (PCA) showed a clear distinction of metabolomic profiles before (CTRL) and after glyphosate (GLY) spray. Most differences were found after the glyphosate spray in both susceptible and resistance plants. PC1 and PC2 explained 30.48% and 9.56% for primary metabolome and 12.59% and 6.94% for secondary, respectively. Susceptible plants displayed more variation overall with GLY treatment, whereas a mixed grouping of Gly-treated resistance plants with controls indicates that resistance plants are affected to different extents, with some plants having minimal effects. Based on the copy number variation and its downstream manifestation on the overall physiology of the plant, we hypothesized that GR biotypes should be distinguishable from GS biotypes based on their native metabolic profile. Our hypothesis was partially supported by the distinct clustering of susceptible biotypes from the resistance biotypes based on their primary and secondary metabolic profile. The distinct clustering was underscored by high abundances of nonaromatic amino acids (arginine, histidine, glutamine), glucose and sugar acids, D-glucaric acid and hexaric acid in the GR-biotypes and ascorbic acid, galactinol, and sugars (glucose and fructose) in the GS-biotypes. These differences were supported by significant differences in the fold change in 19% of the identified primary metabolites. The higher native abundance of shikimate pathway-derived aromatic amino acids in GR-biotypes could be an advantage of higher copies (>60) of EPSPS in a few plants. Interestingly, our analysis revealed a more significant impact of CNVs on the native secondary metabolome. These differences were also significant in the univariate analysis, revealing that 27% of the mass features exhibited variable accumulation. This suggests that CNVs might play a more substantial role in shaping the plant's defense mechanisms and other secondary metabolic pathways. As the metabolic profiles with copy number variation and different treatments can vary and, depending on the effects on each metabolite, could follow complex increasing, decreasing and other varying patterns. To test the association of CNVs with the metabolites, we employed three statistical models: linear regression, non-linear regression, and gene additive model. We compared the results of these models using the Akaike information criterion (AIC) and Bayesian information criterion (BIC) for model selection. Based on AIC and BIC criteria, GAM and, the linear model was the most preferred model for capturing the different metabolic changes with copy number variation. Using GAM model, we predicted 21 and 185 primary mass features in CTRL and GLY treatment respectively, out of 254 significantly (P < 0.05) associated with copy number variation. For the secondary metabolome, there were 785 and 3117 mass features out of 6354 significantly associated with CNVs. From linear model we identified 13 and 185 primary mass features in CTRL and GLY, respectively, significantly (P < 0.05) associated with CNVs and 12 (57.1%) and 180 (97.2%) associated mass features, were common with GAM model. For the secondary metabolome, 665 and 2919 mass features in CTRL and GLY, respectively were associated with CNVs and out of those mass features 585 (88%) and 2801 (96%) were common with GAM model. Overall, the associated metabolites identified from the GAM model were higher, which could decrease the false negative likelihood by considering the complex pattern of metabolic changes. Also, the association of the metabolites in the control treatment with CNVs supported our hypothesis of innate metabolic differences in susceptible and resistant biotypes. Further, we investigated the copy number dosage effect on metabolite concentrations using the GAM model. The observed spread of the half-maximum copy number distribution suggests varying levels of dosage sensitivity that affect metabolite production. Half of their maximum concentration is achieved at a copy number of 41-45 for most primary and secondary metabolites. Few primary metabolites achieved their half-maximal concentration at a lower copy number of 5-10, whereas some secondary metabolites achieved it at a higher copy number of 56-60. Overall, our results indicate a complex interplay between copy number and metabolite production in Palmer amaranth, hence its large spread of glyphosate resistance. We hypothesize that the nonspecific effects of glyphosate extend through different pathways and manifest changes depending on the CNVs. To investigate these pleiotropic effects, first we performed a simple correlation analysis of copy number variation with metabolites of shikimate, TCA pathway and sugars (glucose and sucrose), expected to be major indicators of non-specific effect. We observed a negative correlation between shikimic acid and glucose levels with CNV, while phenylalanine, pyruvic acid, oxalic acid, succinic acid, malic acid, cis-aconitic acid, and sucrose exhibited a positive correlation. The observed negative correlation of CNVs with shikimic acid levels indicates a glyphosate-mediated inhibition of this pathway at a low copy number, whereas normal function at a high copy number. This finding is expected as the shikimate pathway (EPSPS enzyme) is a target of glyphosate; however, changes in the other metabolites not related to shikimate pathways clearly indicate the non-specific pleiotropic effects in response to glyphosate exposure. Further, we investigated the overall perturbation in the primary and secondary metabolome in GLY compared to CTRL in different copy number ranges, including 0-5, 6-20, 21-40, 41-60 and 61-90. Our results indicated an interesting pattern, with perturbation in metabolome decreasing (p-value of significance and Log2 fold change) with increasing copy number. The effects of CNVs percolate through different pathways, as evidenced in the pathway enrichment analysis. In conclusion, our results underscore the complex interplay between EPSPS copy number, glyphosate exposure, and metabolic adaptation.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Kumar R, Tharayil N. Copy number variation facilitated metabolic adaptation introduce robust physiological response of Palmer Amaranth to glyphosate.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Kaur G, Kumar R, Leonard E, Tharayil N. Stress Resilience in WeedsThrough Physiological Priming- A Case Study Using Palmer Amaranth," Weed Science Society ofAmerica, San Antonio Texas, San Antonio, TX, United States. (January 2024
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2024
Citation:
Kumar R, Tharayil N. Metabolomics for deciphering cellular responses of gene dosage variation. Metabolomics Association of North America Conference. Florida October 2024.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2024
Citation:
Kaur G, Kumar R, Leonard E, Tharayil N. Metabolomics as a complementary conduit to elucidate the mechanisms of priming-mediated stress-memory in plants. Metabolomics Association of North America Conference. Florida October 2024.
|
Progress 01/01/22 to 12/31/22
Outputs
Target Audience:The results from the study were conveyed to agriculture producers, educators, academic peers, and students through presentations at various scientific society meetings. Changes/Problems:Flooding in the campus building resulted in a lab closure for 6 months, which adversely affected the research, and multiple ongoing experiments were lost due to the disruption. This significantly compromised the work during 2023. Since then, we have restarted the experiments and are on track to make a recovery in terms of experiments and deliverables. What opportunities for training and professional development has the project provided?Four undergraduate students, two graduate students and one postdoctoral fellow were trained onvarious aspects of the project, including plant cate in the greenhouse, dose-response studies, biochemical analysis, metabolomics, proteomics and transcriptomic, and the date processing of various omics analyses. How have the results been disseminated to communities of interest?The results from various experiments were presented at regional and national scientific meetings, and published in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?1) screen additional amaranthus biotypes for the stress priming experiment. 2) using the data generated in the current year, develop a statisticaland pathway analysis that would predict the mode of action of herbicides based on the metabolomics data.
Impacts
What was accomplished under these goals?
Inducibility of Glyphosate-resistance in Palmer Amaranth: The primary mechanism of glyphosate resistance inA. palmerihas been identified as the amplification of the gene encoding the EPSPS enzyme. However, whether the enhanced EPSPS enzyme activity is constitutively present in biotypes with higher gene copy numbers, or induced after glyphosate application is unknown. A greenhouse study was set up to examinethe inherent physiological and metabolic differences between multiple Palmer amaranth biotypes that have varying degrees of glyphosate susceptibility (GR500.04 to 1.54 kg ae ha-1) as well as the effect of glyphosate on their metabolic activity. We hypothesize that, although increased EPSPS copy number is the primary means of glyphosate resistance mechanism in GR Palmer amaranth, the overall phytochemical profile, and thus potentially the antioxidant activities, across the biotypes will show a positive correlation with their respective GR50. Metabolite profiling showed that although the total abundance of the various metabolic classes (amino acids, sugars, organic acids) was similar across the biotypes, significant compositional differences existed among them. Comparing the most resistant (C1B1) biotype with the most susceptible (C3) biotype, it was observed that the C1B1 biotype was innately abundant in metabolites derived from the phenylpropanoid pathway. Furthermore, the antioxidant capacity of the R-biotypes was inherently higher, which increased with increasing GR50. Following glyphosate application, the S-biotypes had a significant increase in the total amino acid coupled with a decrease in phenolics and flavonoid content. In contrast, the relative abundance of most metabolites in the R-biotypes either remained the same or increased. Among the R-biotypes, C1B1 significantly increased its flavonoid content, but this did not result in an increased anti-oxidant capacity. On the other hand, although the C3 biotype had a decrease in its flavonoid concentration, it increased its total antioxidant capacity following glyphosate application. These results indicate that the phytochemistry and the antioxidant capacity that might play a complementary role in glyphosate resistance are partly induced after glyphosate application rather than being constitutively expressed. Taken together, the results in this study indicated that in the absence of stress, the phytochemical profiles between the S- and R-biotypes are comparable. However, the R-biotypes had inherently high anti-oxidant potential. When exposed to glyphosate stress, to survive, the R-biotypes adopted different physiological and metabolic strategies, including increasing antioxidants and metabolite content coupled with the innately higher anti-oxidant capacity, to respond to the glyphosate-induced oxidative stress. In contrast, after glyphosate treatment, the content of total phenolic and flavonoids decreases in S-biotypes. Thus, these results indicate that the phytochemistry and the antioxidant capacity might play a complementary role in glyphosate resistance observed in amaranth, and are partly induced after glyphosate application, rather than being constitutively expressed. Screening of Palmer Amaranth populations for resistance and stress priming. We studied the priming-induced stress resilience in Palmer amaranth (Amaranthus palmeri) biotypes resistant to glyphosate due to its potential ability to enhance phytochemical production from EPSPS gene amplification. A total of 72 palmer amaranth populations were collected from five states in the southern United States. The levels of glyphosate resistance were documented in these biotypes via dose-response assays, and EPSPS copy numbers were determined. The EPSPS copy numbers varied between 1 and 160 across these biotypes, and 50 of these biotypes exhibited varying degrees of resistance to glyphosate. A subset of biotypes was further tested for hormesis-mediate priming response to herbicide stress. Four-week-old amaranth plants were primed with a sublethal dose of glyphosate (40 times lower than the GR50) and subsequently subjected to trigger stress doses (1x, 2x, and 3x of the prime concertation) after the initial metabolic perturbations subsided. During the priming phase, the cellular perturbation from glyphosate was evident from the immediate accumulation of shikimate, which subsequently decreased and stabilized by 72hrs. During the trigger phase, the shikimate accumulation positively correlated with glyphosate concentrations in the non-primed plants, whereas the shikimate content in the primed plants did not increase with increasing glyphosate concentration. The changes in pool size of > 4500 mass features captured using the global metabolomics approach also reflected the resilience conferred by the sublethal dose of glyphosate, where the glyphosate trigger perturbed the metabolic pool of the glyphosate-primed plants to a lesser degree. Omics studies to elucidate the cellular physiology (continued): Physiological stress response in plants results from modulations at genomic, transcriptomic, proteomic, and metabolomic levels. The degree to which herbicide-induced simultaneous perturbation in these levels largely remains unknown. This study, continuing from last year, aimed to dissect the multi-level stress response induced by glyphosate, the most widely used herbicide in the world, in glyphosate-resistant (GR) and -susceptible (GS) biotype of a dominant agricultural weed, Palmer amaranth. Analysis of leaf tissues twenty-four hours after glyphosate treatment captured changes in 30371 transcripts, 5606 proteins, 80 primary, and 525 secondary metabolites. This year, to further explore the association between these different levels of molecular activities in GR and GS biotypes with glyphosate treatment, we selected all the differentially expressed genes, proteins, and metabolites for integrated analysis in paintomics. The major integrated, enriched pathways in the GR-biotypes were phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, metabolism-raffinose, Glutathione metabolism, and metabolism2nd-Flavonoids. The notable pathways affected in GS-biotype were photosynthesis, biosynthesis of secondary metabolites, carbon fixation, ribosome, metabolism, and synthesis-JA. Further, based on this metabolite hub analysis, Ferulic acid was the major regulated metabolic feature in the GR-biotype, whereas amino acids and sugars were in the GS-biotype. Further metabolite class activity analysis, which tests if the measured metabolite class has a high proportion of compounds with significant changes, identified amino acids as the significant perturbed metabolite class in GS-biotype only. Apart from its role in aromatic amino acid synthesis, the shikimate pathway, a major target of Glyphosate, also acts as a major sink for carbon intermediates. Since carbon metabolism is perturbed in the GS-biotypes in response to Glyphosate treatment, it could also result in system-wide perturbations, including changes in the non-aromatic amino acids;metabolite abundance with transcripts or proteins was minimal, especially for secondary metabolites. This indicates that non-lethal doses of herbicide have the potential to prime the GR-biotype against future stressors. The glyphosate-induced inhibition of basal metabolic pathways, including photosynthesis and central carbon metabolism at transcript and metabolite levels, was observed only in GS-biotype, demonstrating the upregulation of stress response systems in GR-biotype could occur without the growth penalty detected in GS-biotype. Overall, our study highlights the non-specificity of the herbicide-induced cellular level perturbations in A. palmeri and suggests that the secondary effects might prime plants to future biotic/abiotic stressors.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Sandhu P, Leonard E, Nandula V, Saski C, McMahan C, Tharayil N. Multi-omics analysis of glyphosate-induced response in Palmer amaranth (Amaranthus palmeri) highlights induced upregulation of stress response systems in herbicide-resistant biotypes. (in review)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Maroli, A, Tharayil, N. Metabolic adaptations and anti-oxidant responses to glyphosate induced oxidative stress in Palmer amaranth is dependent on the level of glyphosate resistance. (In review)
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Progress 01/01/21 to 12/31/21
Outputs
Target Audience:The results from the study were conveyed to agriculture producers, educators, academic peers, and students through presentations at various scientific society meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One MSstudent, one postdoctoral scholar, and one research assistant (all females) was trained on various aspects of the project. The project also provided learning opportunity to one undergraduate student. M.S. student successfully graduated. How have the results been disseminated to communities of interest?The results from the study was been presented at six local/regional/national meetings. Two manuscripts from the project are currently under review. What do you plan to do during the next reporting period to accomplish the goals? Elucidate stress priming in amaranth from a metabolic/proteomic and transcriptomic level Use metabolomics as a novel tool to discern the mode of action of herbicide. Widen the biotypes used for the study by collecting and screening more biotypes of glyphosate-resistant amaranth
Impacts
What was accomplished under these goals?
PROJECT 1: The evolution of resistance to herbicides is a major threat to global agricultural production systems. To date, 55 weed species have developed resistance against glyphosate, the most widely used herbicide worldwide. Palmer amaranth (Amaranthus palmeri) is a dominant glyphosate-resistant weed that causes more than 50% yield loss in row crop production systems of the southeastern US. The primary mechanism of resistance in Palmer amaranth is gene amplification of the target enzyme, 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, the glyphosate-induced cellular physiology of glyphosate-resistant (GR) and -susceptible (GS) biotypes remains obscure, especially since the partial blockage of the shikimate pathway is evident in GR biotypes immediately after glyphosate application. We integrated transcriptomic, proteomic, and metabolomic approaches to elucidate the glyphosate-induced stress perturbations in GR and GS-biotypes of Palmer amaranth. RNAseq transcriptomics, shot-gun proteomics, and untargeted metabolomics analysis of leaf tissues twenty-four hours after glyphosate treatment resulted in 30371 transcripts, 5606 master proteins, 80 primary, and 525 secondary metabolites, respectively. Correlation analysis revealed a positive association (r = 0.51) between the transcripts and the corresponding protein levels across biotypes in glyphosate-treated tissues. Functional analysis showed upregulation of ABA-activated signaling pathway, signaling receptor activity, and protein phosphatase inhibitor activity (GO terms) and glutathione metabolism (KEGG pathway) across biotypes at both transcript and protein levels. On a differential expression basis, the effect of glyphosate was more significant on GS-biotype compared to GR-biotype at transcript (3349 vs 1028), protein (226 vs 71) and primary metabolite (19 vs 6). Interestingly, the secondary metabolome was more perturbed in GR-biotype (120 vs 68). Glyphosate-induced reduction of identified phenylpropanoids was similar across the biotypes. However, there was a significant decrease in the abundance of triterpenoids in the GR-biotype that was not observed in the GS-biotype. Overall This study lays the groundwork for integrating multiple omics techniques to elucidate the cellular physiology of herbicide perturbations. PROJECT 2: Modifications of the phytochemical profile form a vital component of physiological stress adaptation in plants. However, the specificity and uniqueness of phytochemical changes with respect to the identity of stressors is less known. We investigated the commonality and specificity of metabolic perturbations induced by a specific stressor - glyphosate, and a general stressor - drought, across multiple glyphosate-resistant (GR) and -susceptible (GS) biotypes of a dominant agricultural weed, Amaranthus palmeri. In the absence of stress, the native metabolite profiles of GS- and GR- biotypes was similar, and amplification of the EPSPS gene in GR-biotypes did not translate to a higher abundance of downstream metabolites. Further, glyphosate treatment initially inhibited the shikimate pathway in both GS- and GR-biotypes, from which the GR biotype recovered, indicating inducibility in the functionalization of EPSPS enzyme. The accumulation of phenylpropanoids produced downstream of shikimate pathway, was higher in GR-biotypes than GS-biotypes, with a preferential accumulation of compounds with higher antioxidant potential. However, this increase was not observed in response to drought treatment, where the metabolic perturbations were pervasive, but limited, in magnitude compared to glyphosate stress. Overall, while native phytochemistry of A. palmeri was similar irrespective of the level of glyphosate susceptibility, the specific stressor, glyphosate, imparted metabolic perturbations that were localized but higher in magnitude, while the specificity of phytochemical response to the general stressor, drought, was minimal. Taken together, these results suggest that, at the metabolic level, the glyphosate resistance mechanism in A. palmeri is partly induced and specific to herbicide stress.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Sandhu P, Leonard E, Nandula V, Tharayil N. Metabolite profiling across populations of Palmer amaranth (Amaranthus palmeri) indicates specificity and inducibility of phytochemical response to glyphosate stress. The Plant Journal
- Type:
Theses/Dissertations
Status:
Submitted
Year Published:
2022
Citation:
Sandhu, P. 2022. Elucidating the Cellular Physiology of Glyphosate Resistance in Palmer Amaranth (Amaranthus palmeri) using Integrated Omics Approaches. MS Thesis. Clemson University Clemson SC.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Sandhu P, Leonard E, Nandula V, Saski C, McMahan C, Tharayil N. Multi-omics analysis of glyphosate-induced response in Palmer amaranth (Amaranthus palmeri) highlights induced upregulation of stress response systems in herbicide-resistant biotypes. bioRxiv
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu PK et al. 2021, Global Metabolomics elucidates the Differential Cellular-level Physiology of Specific vs General Stressors in Plants. Clemson Biological Sciences Annual Student Symposium (CBASS), Online, March 27, 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu PK et al. 2021, Integration of Proteomics and Metabolomics approaches to elucidate the Glyphosate-Induced Stress Response in Amaranthus palmeri. 82nd Meeting of Southern Section of the American Society of Plant Biologists, Online, April 16 18, 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu PK et al. 2021, Systems-level View of Glyphosate-Induced Stress Response in Amaranthus palmeri through Integration of Proteomics, Metabolomics and Transcriptomics approaches. Plant Biology Worldwide Summit, Online, July 19 23, 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu PK et al. 2021, Mapping the cellular physiology of glyphosate resistance in Palmer amaranth using global metabolomic approaches. 3rd annual Metabolomics Association of North America conference (MANA 2021), Online, October 18 21, 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Sandhu PK (2021), What does not kill you, makes you stronger . Clemson 3-Minute Thesis, Clemson University, November 19, 2021
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