Open Access
Unravelling the phytotoxic effects of glyphosate on sensitive and resistant Amaranthus Palmeri populations by GC-MS and LC-MS metabolic profiling
(MDPI, 2023) Zulet González, Ainhoa; Gorzolka, Karin; Döll, Stefanie; Gil Monreal, Miriam; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Glyphosate, the most successful herbicide in history, specifically inhibits the activity of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), one of the key enzymes in the shikimate pathway. Amaranthus palmeri is a driver weed in agriculture today that has evolved glyphosate-resistance through increased EPSPS gene copy number and other mechanisms. Non-targeted GC–MS and LC–MS metabolomic profiling was conducted to examine the innate physiology and the glyphosate-induced perturbations in one sensitive and one resistant (by EPSPS amplification) population of A. palmeri. In the absence of glyphosate treatment, the metabolic profile of both populations was very similar. The comparison between the effects of sublethal and lethal doses on sensitive and resistant populations suggests that lethality of the herbicide is associated with an amino acid pool imbalance and accumulation of the metabolites of the shikimate pathway upstream from EPSPS. Ferulic acid and its derivatives were accumulated in treated plants of both populations, while quercetin and its derivative contents were only lower in the resistant plants treated with glyphosate.
Open Access
Role of oxidative stress in the physiology of sensitive and resistant Amaranthus palmeri populations treated with herbicides inhibiting acetolactate synthase
(Frontiers Media, 2023) Eceiza, Mikel Vicente; Barco Antoñanzas, María; Gil Monreal, Miriam; Huybrechts, Michiel; Zabalza Aznárez, Ana; Cuypers, Ann; Royuela Hernando, Mercedes; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The aim of the present study was to elucidate the role of oxidative stress in the mode of action of acetolactate synthase (ALS) inhibiting herbicides. Two populations of Amaranthus palmeri S. Watson from Spain (sensitive and resistant to nicosulfuron, due to mutated ALS) were grown hydroponically and treated with different rates of the ALS inhibitor nicosulfuron (one time and three times the field recommended rate). Seven days later, various oxidative stress markers were measured in the leaves: H2O2, MDA, ascorbate and glutathione contents, antioxidant enzyme activities and gene expression levels. Under control conditions, most of the analysed parameters were very similar between sensitive and resistant plants, meaning that resistance is not accompanied by a different basal oxidative metabolism. Nicosulfuron-treated sensitive plants died after a few weeks, while the resistant ones survived, independently of the rate. Seven days after herbicide application, the sensitive plants that had received the highest nicosulfuron rate showed an increase in H2O2 content, lipid peroxidation and antioxidant enzymatic activities, while resistant plants did not show these responses, meaning that oxidative stress is linked to ALS inhibition. A supralethal nicosulfuron rate was needed to induce a significant oxidative stress response in the sensitive population, providing evidence that the lethality elicited by ALS inhibitors is not entirely dependent on oxidative stress.
Open Access
Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots
(Wiley, 2016) Armendáriz García, Óscar; Gil Monreal, Miriam; Zulet González, Amaia; Zabalza Aznárez, Ana; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis-inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched-chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less-efficient, ATP-producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.
Open Access
Cysteine proteases are activated in sensitive Amaranthus palmeri populations upon treatment with herbicides inhibiting amino acid biosynthesis
(Wiley, 2023) Barco Antoñanzas, María; Font Farre, María; Eceiza, Mikel Vicente; Gil Monreal, Miriam; Van der Hoorn, Reiner; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The herbicides glyphosate and pyrithiobac inhibit the enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS) in the aromatic amino acid biosynthetic pathway and acetolactate synthase (ALS) in the branched-chain amino acid biosynthetic pathway, respectively. Here we characterise the protease activity profiles of a sensitive (S), a glyphosate-resistant (GR) and a multiple-resistant (MR) population of Amaranthus palmeri in response to glyphosate and pyrithiobac. Amino acid accumulation and cysteine protease activities were induced with both herbicides in the S population and with pyrithiobac in the GR population, suggesting that the increase in cysteine proteases is responsible for the increased degradation of the available proteins and the observed increase in free amino acids. Herbicides did not induce any changes in the proteolytic activities in the populations with target-site resistance, indicating that this effect was only induced in sensitive plants.
Open Access
Increased glyphosate-induced gene expression in the shikimate pathway is abolished in the presence of aromatic amino acids and mimicked by shikimate
(Frontiers Media, 2020) Zulet González, Ainhoa; Barco Antoñanzas, María; Gil Monreal, Miriam; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The herbicide glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid (AAA) biosynthetic pathway, also known as the shikimate pathway. Amaranthus palmeri is a fast-growing weed, and several populations have evolved resistance to glyphosate through increased EPSPS gene copy number. The main objective of this study was to elucidate the regulation of the shikimate pathway and determine whether the regulatory mechanisms of glyphosate-sensitive and glyphosate-resistant plants were different. Leaf disks of sensitive and resistant (due to EPSPS gene amplification) A. palmeri plants were incubated for 24 h with glyphosate, AAA, glyphosate + AAA, or several intermediates of the pathway: shikimate, quinate, chorismate and anthranilate. In the sensitive population, glyphosate induced shikimate accumulation and induced the gene expression of the shikimate pathway. While AAA alone did not elicit any change, AAA applied with glyphosate abolished the effects of the herbicide on gene expression. It was not possible to fully mimic the effect of glyphosate by incubation with any of the intermediates, but shikimate was the intermediate that induced the highest increase (three-fold) in the expression level of the genes of the shikimate pathway of the sensitive population. These results suggest that, in this population, the lack of end products (AAA) of the shikimate pathway and shikimate accumulation would be the signals inducing gene expression in the AAA pathway after glyphosate application. In general, the effects on gene expression detected after the application of the intermediates were more severe in the sensitive population than in the resistant population. These results suggest that when EPSPS is overexpressed, as in the resistant population, the regulatory mechanisms of the AAA pathway are disrupted or buffered. The mechanisms underlying this behavior remain to be elucidated.
Open Access
Physiological approach to the use of the natural compound quinate in the control of sensitive and resistant Papaver rhoeas
(MDPI, 2020) Zabalza Aznárez, Ana; Zulet González, Ainhoa; Barco Antoñanzas, María; Eceiza, Mikel Vicente; Gil Monreal, Miriam; Royuela Hernando, Mercedes; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Quinate (1,3,4,5-tetrahydroxycyclohexanecarboxylate) is a compound synthesized in plants through a side-branch of the shikimate biosynthesis pathway, which is accumulated after glyphosate and acetolactate synthase inhibiting herbicides (ALS-inhibitors) and has phytotoxic potential. The objective of this study was to evaluate the phytotoxicity of quinate on several weed species. Among the species evaluated, Cynodon dactylon, Bromus diandrus, Lolium rigidum, Sinapis alba, and Papaver rhoeas, P. rhoeas was the most sensitive, and its growth was controlled with quinate concentrations above 100 mM at the phenological stage of 6–8 true leaves. A physiological study, including the shikimate pathway and the physiological markers of ALS-inhibitors (carbohydrates and amino acids), was performed in the sensitive and resistant plants treated with sulfonylureas or quinate. The typical physiological effects of ALS-inhibitors were detected in the sensitive population (free amino acid and carbohydrate accumulation) and not detected in the resistant population. The mode of action of quinate appeared to be related to general perturbations in their carbon/nitrogen metabolism rather than to specific changes in the shikimate pathway. These results suggest the possibility of using quinate in the weed control management of P. rhoeas.
Open Access
Characterization of the Amaranthus palmeri physiological response to glyphosate in susceptible and resistant populations
(American Chemical Society, 2016) Fernández Escalada, Manuel; Gil Monreal, Miriam; Zabalza Aznárez, Ana; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
The herbicide glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS) in the aromatic amino acid (AAA) biosynthetic pathway. The physiologies of an Amaranthus palmeri population exhibiting resistance to glyphosate by EPSPS gene amplification (NC-R) and a susceptible population (NC-S) were compared. The EPSPS copy number of NC-R plants was 47.5-fold the copy number of NC-S plants. Although the amounts of EPSPS protein and activity were higher in NC-R plants than in NC-S plants, the AAA concentrations were similar. The increases in total free amino acid and in AAA contents induced by glyphosate were more evident in NC-S plants. In both populations, the EPSPS protein increased after glyphosate exposure, suggesting regulation of gene expression. EPSPS activity seems tightly controlled in vivo. Carbohydrate accumulation and a slight induction of ethanol fermentation were detected in both populations.
Open Access
ERF-VII transcription factors induce ethanol fermentation in response to amino acid biosynthesis-inhibiting herbicides
(Oxford University Press, 2019) Gil Monreal, Miriam; Giuntoli, Beatrice; Zabalza Aznárez, Ana; Licausi, Francesco; Royuela Hernando, Mercedes; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Herbicides inhibiting either aromatic or branched-chain amino acid biosynthesis trigger similar physiological responses in plants, despite their different mechanism of action. Both types of herbicides are known to activate ethanol fermentation by inducing the expression of fermentative genes; however, the mechanism of such transcriptional regulation has not been investigated so far. In plants exposed to low-oxygen conditions, ethanol fermentation is transcriptionally controlled by the ethylene response factors-VII (ERF-VIIs), whose stability is controlled in an oxygen-dependent manner by the Cys-Arg branch of the N-degron pathway. In this study, we investigated the role of ERF-VIIs in the regulation of the ethanol fermentation pathway in herbicide-treated Arabidopsis plants grown under aerobic conditions. Our results demonstrate that these transcriptional regulators are stabilized in response to herbicide treatment and are required for ethanol fermentation in these conditions. We also observed that mutants with reduced fermentative potential exhibit higher sensitivity to herbicide treatments, thus revealing the existence of a mechanism that mimics oxygen deprivation to activate metabolic pathways that enhance herbicide tolerance. We speculate that this signaling pathway may represent a potential target in agriculture to affect tolerance to herbicides that inhibit amino acid biosynthesis.
Open Access
Quinate-enhanced glyphosate toxicity is related to the accumulation of quinate derivatives
(Elsevier, 2024) Zulet González, Ainhoa; Gil Monreal, Miriam; Gorzolka, Karin; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Institute for Multidisciplinary Research in Applied Biology - IMAB
Glyphosate is the most widely used herbicide and works by inhibiting the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) of the shikimate pathway, preventing the aromatic amino acid biosynthesis. When applied to plants, it provokes the accumulation of quinate, a metabolite synthesized through a side branch of the shikimate pathway. The joint application of glyphosate and quinate enhanced glyphosate efficacy on Amaranthus palmeri, requiring one-quarter of the recommended dose of glyphosate for complete control. Expression of the genes of the shikimate pathway and non-targeted GC-MS metabolic profiling were conducted to compare the physiological response after glyphosate, quinate or the combination of both. A perturbed gene expression of the shikimate pathway was detected after quinate applied alone, while no relevant changes in the metabolome were detected. The sub-lethal glyphosate treatment induced gene expression in the shikimate pathway, accumulation of the metabolites located upstream EPSPS and disturbances in the amino acid content. The exacerbation of the phytotoxicity in the lethal combined treatment was not related to any specific change in the expression level of the shikimate pathway. Metabolic profiling indicated that the accumulation of quinate and quinate derivatives detected after quinate applied alone was severely enhanced after the combined treatment of quinate and glyphosate.
Open Access
Efecto del glifosato en la expresión génica de la ruta del siquimato en Amaranthus palmeri
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2017) Fernández Escalada, Manuel; Zulet González, Ainhoa; Gil Monreal, Miriam; Zabalza Aznárez, Ana; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Este estudio ha abordado el efecto del herbicida glifosato en la regulación de la ruta del siquimato en la especie Amaranthus palmeri, comparando una población sensible a glifosato con una población resistente. Se han determinado, mediante PCR cuantitativa (qPCR), el efecto de dos dosis de glifosato sobre la expresión génica de los enzimas de la ruta del siquimato. También se ha determinado la actividad enzimática de los enzimas post-corismato antranilato sintasa (AS) y corismato mutasa (CM). Se ha podido observar que el gran aumento en el número de copias génicas del enzima EPSPS en la población resistente no tiene un efecto pleiotrópico basal en la expresión de los demás genes de esta ruta de síntesis de los aminoácidos aromáticos. Tras el tratamiento con glifosato, se observó, en ambas poblaciones, una inducción general de la expresión de los genes de la ruta del siquimato, dependiente de la dosis de glifosato. Es destacable que la aplicación del glifosato provoca el aumento de la expresión y actividad AS, y la tendencia opuesta en expresión CM, lo que conlleva un flujo preferente hacia la formación de triptófano en lugar de tirosina y fenilalanina.