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
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
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
New insights in the regulation of the shikimate pathway after quinate as plyphosate enhancer in Amaranthus palmeri
(2020) Zulet González, Ainhoa; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Ciencias; Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua
El glifosato es el herbicida más empleado a nivel mundial. Su mecanismo de acción es la inhibición de la enzima 5‐enolpiruvilsiquimato‐3‐fosfato sintasa (EPSPS), enzima clave dentro de la ruta de biosíntesis de los amino ácidos aromáticos, también conocida como la ruta del siquimato. Uno de los procesos fisiológicos descritos tras la aplicación de este herbicida es la acumulación de quinato, un metabolito secundario, formado a partir de una ramificación lateral de la ruta del siquimato. Tras detectar efectos fisiológicos por la aplicación de quinato exógeno de forma aislada, se planteó la posibilidad de utilización de este compuesto como potenciador del glifosato para reducir las dosis de herbicida en el control de la mala hierba Amaranthus palmeri. En esta especie se han desarrollado poblaciones resistentes a glifosato, entre las cuales se encuentra el mecanismo de resistencia de sobreexpresión del gen EPSPS, lo que lleva a una sobreexpresión del enzima diana del herbicida. El principal objetivo de esta tesis fue el de profundizar en las alteraciones de la ruta del siquimato por el herbicida glifosato y el uso del quinato como potenciador de este herbicida en el control de plantas de A. palmeri sensibles y resistentes a glifosato (por amplificación génica). En este trabajo se determinó que la aplicación de quinato un día después del glifosato (tratamiento combinado) convertía en letal una dosis sub‐letal de herbicida en la población sensible. Estos resultados abren la posibilidad de aplicar ambos compuestos para mejorar la eficacia del herbicida y de reducir las dosis a aplicar en el control de poblaciones sensibles. La mayor toxicidad del tratamiento combinado estuvo relacionada con la exacerbación en los marcadores fisiológicos previamente descritos para este herbicida (acumulación del contenido de siquimato y de aminoácidos libres). Se estudió el comportamiento de la ruta del siquimato tras el tratamiento combinado a nivel metabolómico y transcriptómico, tratando de encontrar respuesta al incremento de eficacia observado en el tratamiento combinado. Los resultados obtenidos parecen indicar que el incremento de la toxicidad del tratamiento combinado estaría relacionado con cambios a nivel metabólico, debido en concreto a un incremento en el contenido de los derivados del quinato, y no debido a cambios de niveles de transcripción. Se realizó un perfil metabólico no dirigido para comparar el perfil metabólico de ambas poblaciones tratadas con glifosato y para determinar si otros cambios en el perfil metabólico podrían estar contribuyendo al incremento en la toxicidad del tratamiento combinado. Entre todos los compuestos del metabolismo primario y secundario evaluados, los derivados del quinato fueron los únicos compuestos que se acumularon en este tratamiento de manera diferencial, evidenciando su papel en el incremento de la toxicidad de este tratamiento. Por último, se evaluó el papel de los amino ácidos aromáticos y de algunos intermediarios metabólicos (siquimato, quinato, corismato y antranilato) en la regulación de la ruta del siquimato mediante incubación de discos de hojas con ellos. Se observó que los aminoácidos aromáticos, al ser aplicados en combinación con el herbicida, neutralizaron los efectos del glifosato a nivel transcipcional pero no la acumulación del siquimato. Ninguno de los intermediarios metabólicos evaluados mimetizó completamente el efecto del glifosato en la ruta del siquimato. Sin embargo, el siquimato fue el metabolito que indujo una sobreexpresión de la mayoría de los genes de la ruta del siquimato, de una manera similar al herbicida. Estos resultados sugieren que la inducción en la transcripción observada tras el tratamiento con glifosato podría estar mediada, al menos en parte, por la acumulación de siquimato. Resumiendo, esta tesis aporta nuevos aspectos en la regulación de la ruta del siquimato tras la aplicación de glifosato, y aborda el efecto del tratamiento combinado del herbicida con quinato a nivel práctico y fisiológico, tratando de encontrar las causas de ese incremento en la toxicidad.
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.