Open Access
Primary metabolism in an Amaranthus palmeri population with multiple resistance to glyphosate and pyrithiobac herbicides
(Elsevier, 2022) Barco Antoñanzas, María; Gil Monreal, Miriam; Eceiza, Mikel Vicente; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, PJUPNA20-6138
The objective of this work was to characterize the resistance mechanisms and the primary metabolism of a multiple resistant (MR) population of Amaranthus palmeri to glyphosate and to the acetolactate synthase (ALS) inhibitor pyrithiobac. All MR plants analysed were glyphosate-resistant due to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene amplification. Resistance to pyrithiobac was more variable among individuals and was related to point mutations at five positions in the ALS gene sequence: A122, A205, W574, S653 and G654. All MR plants were heterozygous for W574, the most abundant mutation. In nontreated plants, the presence of mutations did not affect ALS functionality, and plants with the W574L mutation showed the highest ALS resistance level to pyrithiobac. The accumulation of the transcripts corresponding to several genes of the aromatic amino acid (AAA) and branched-chain amino acid (BCAA) pathways detected in nontreated MR plants indicated additional effects of EPSPS gene amplification and ALS mutations. The physiological performance of the MR population after treatment with glyphosate and/or pyrithiobac was compared with that of a sensitive (S) population. The increase induced in total soluble sugars, AAA or BCAA content by both herbicides was higher in the S population than in the MR population. Physiological effects were not exacerbated after the mixture of both herbicides in S or in MR populations. This study provides new insights into the physiology of a multiple resistant A. palmeri, which could be very useful for achieving effective management of this weed.
Open Access
Fermentation and alternative oxidase contribute to the action of amino acid biosynthesis-inhibiting herbicides
(Elsevier, 2015) Zulet González, Amaia; Gil Monreal, Miriam; Zabalza Aznárez, Ana; Dongen, Joost T. van; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Acetolactate synthase inhibitors (ALS-inhibitors) and glyphosate (GLP) are two classes of herbicide that act by the specific inhibition of an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. The physiological effects that are detected after application of these two classes of herbicides are not fully understood in relation to the primary biochemical target inhibition, although they have been well documented. Interestingly, the two herbicides’ toxicity includes some common physiological effects suggesting that they kill the treated plants by a similar pattern despite targeting different enzymes. The induction of aerobic ethanol fermentation and alternative oxidase (AOX) are two examples of these common effects. The objective of this work was to gain further insight into the role of fermentation and AOX induction in the toxic consequences of ALS-inhibitors and GLP. For this, Arabidopsis T-DNA knockout mutants of alcohol dehydrogenase (ADH) 1 and AOX1a were used. The results found in wild-type indicate that both GLP and ALS-inhibitors reduce ATP production by inducing fermentation and alternative respiration. The main physiological effects in the process of herbicide activity upon treated plants were accumulation of carbohydrates and total free amino acids. The effects of the herbicides on these parameters were less pronounced in mutants compared to wild-type plants. The role of fermentation and AOX regarding pyruvate availability is also discussed.
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
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
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.
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 y de diferentes intermediarios metabólicos en la regulación de la ruta del siquimato
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2017) Zulet González, Ainhoa; 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
En este estudio se han abordado los mecanismos de regulación de la ruta del siquimato en la biosíntesis de los aminoácidos aromáticos. Para ello se han comparado el contenido de siquimato y de los enzimas EPSPS (5-enolpiruvilsiquimato- 3-fosfato sintasa) y DAHPS (3-Deoxi-D-arabino-heptulosonato-7-fosfato sintasa, enzima de entrada a la ruta) en hojas de dos poblaciones de Amaranthus palmeri (una sensible y otra resistente al glifosato) incubadas con glifosato y/o con diferentes intermediarios de la ruta (siquimato, quinato, fosfoenolpiruvato, y aminoácidos aromáticos). En ambas poblaciones se dieron similares efectos regulatorios de los intermediarios, evidenciando que la sobreexpresión de EPSPS no modifica de manera significativa la regulación general de la ruta. El enzima DAHPS se confirma como enzima clave en la regulación y en la respuesta al glifosato; su síntesis es inhibida por los productos finales de la ruta y por el quinato, y su regulación puede explicar la acumulación de siquimato tras la aplicación de glifosato.
Open Access
La sobreexpresión del gen ALDH7B4 alivia los efectos provocados por herbicidas inhibidores de la biosíntesis de aminoácidos
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2017) Gil Monreal, Miriam; Zabalza Aznárez, Ana; Missihoun, Tagnon D.; Bartels, Dorothea; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
En este trabajo se ha profundizado en el conocimiento de los efectos fisiológicos provocados por los herbicidas imazamox y glifosato. En concreto, se ha estudiado el papel de la ALDH7B4, aldehído deshidrogenasa, que contribuye a la tolerancia de las plantas a diversos estreses. Se cultivaron plantas de Arabidopsis thaliana silvestres y plantas que sobreexpresan el gen ALDH7B4, que fueron tratadas con dosis comparables de imazamox o glifosato. Se observó que ambos genotipos presentan valores similares de malondialdehído, indicando que la ALDH7B4 no participa en la detoxificación de aldehídos derivados de la peroxidación lipídica. Por otro lado, se monitorizaron los principales efectos fisiológicos provocados por este tipo de herbicidas y se observó que los efectos de los herbicidas (contenido en carbohidratos y crecimiento) se atenúan en las plantas mutantes, indicando que la ALDH7B4 contribuye a aliviar los efectos provocados por estos herbicidas.
Open Access
Physiological performance of glyphosate and imazamox mixtures on Amaranthus palmeri sensitive and resistant to glyphosate
(Nature Research, 2019) Fernández Escalada, Manuel; Zulet González, Ainhoa; 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 herbicides glyphosate and imazamox inhibit the biosynthetic pathway of aromatic amino acids (AAA) and branched-chain amino acids (BCAA), respectively. Both herbicides share several physiological effects in the processes triggered in plants after herbicide application that kills the plant, and mixtures of both herbicides are being used. The aim of this study was to evaluate the physiological effects in the mixture of glyphosate and imazamox in glyphosate-sensitive (GS) and -resistant (GR) populations of the troublesome weed Amaranthus palmeri. The changes detected in the physiological parameters after herbicide mixtures application were similar and even less to the changes detected after individual treatments. This pattern was detected in shikimate, amino acid and carbohydrate content, and it was independent of the EPSPS copy number, as it was detected in both populations. In the case of the transcriptional pattern of the AAA pathway after glyphosate, interesting and contrary interactions with imazamox treatment were detected for both populations; enhancement of the effect in the GS population and alleviation in the GR population. At the transcriptional level, no cross regulation between AAA and BCAA inhibitors was confirmed. This study suggests that mixtures are equally or less toxic than herbicides alone, and would implicate careful considerations when applying the herbicide mixtures.
Open Access
The moderate oxidative stress induced by glyphosate is not detected in Amaranthus palmeri plants overexpressing EPSPS
(Elsevier, 2022) Gil Monreal, Miriam; Barco Antoñanzas, María; Zabalza Aznárez, Ana; Royuela Hernando, Mercedes; Eceiza, Mikel Vicente; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, PJUPNA20-6138
The present study aimed to determine whether glyphosate-induced oxidative stress is directly related to the action mechanism of this herbicide (5-enolpyruvylshikimate-3-phosphate synthase or EPSPS inhibition) and analyse the role of oxidative stress in glyphosate toxicity of the weed Amaranthus palmeri S. Wats. Two kinds of populations were studied using EPSPS amplification: glyphosate-sensitive and glyphosate-resistant (by gene amplification). Plants were grown hydroponically and treated with different glyphosate doses, after which several oxidative stress markers were measured in the leaves. Untreated, sensitive and resistant plants showed similar values for the analysed parameters. Treated glyphosate-sensitive plants showed an increase in shikimate, superoxide and H2O2 contents and dose-dependent lipid peroxidation and antioxidant responses; however, none of these effects were observed in resistant plants, indicating that glyphosate-induced oxidative stress is related to EPSPS inhibition. Oxidative stress is associated with an increase in the activity of peroxidases due to EPSPS inhibition, although the link between both processes remains elusive. The fact that some glyphosate doses were lethal but did not induce major oxidative damage provides evidence that glyphosate toxicity is independent of oxidative stress.