BUSCADOR RECOLECTA

Datos de investigación alojados en: https://doi.org/10.5281/zenodo.16091477, 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., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R) and Spanish Ministry of Science and Innovation (2020 117723-RB-100). A. Z-G received funding from fellowship through Universidad Pública de Navarra.

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., MGM received funding from fellowships through Universidad Pública de Navarra. This work was financially supported by a grant from the Ministerio Español de Economía y Competitividad (AGL-2016-77531R).

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., This work was supported by two grants from the Ministerio Español de Economía y Competitividad (Project number AGL2016-77531-R). AZ-G and MB-A received funding from fellowships through Universidad Pública de Navarra.

Durante el siglo pasado, la producción agrícola se ha ido incrementando en todo el mundo gracias a la mejora en sus técnicas de manejo. Una de las herramientas más poderosas para alcanzar ese incremento en la producción fueron los herbicidas, los cuales eliminan las malas hierbas que compiten con los cultivos por los recursos naturales. Sin embargo, el uso inapropiado de los herbicidas ha desencadenado la aparición de resistencias en malas hierbas, un problema global para la agricultura que se ha ido incrementando durante los últimos 30 años. El glifosato es el herbicida más usado en todo el mundo debido a su efectividad como herbicida total, y sobre todo desde la aparición de cultivos genéticamente modificados resistentes a este herbicida. La diana del glifosato es la enzima 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), en la ruta biosintética de los aminoácidos aromáticos (AAA). Una de las peores malas hierbas que se ha descrito como resistente al glifosato es Amaranthus palmeri, cuyo mecanismo de resistencia es la amplificación génica de la enzima EPSPS, lo que conlleva una sobreexpresión de ésta enzima diana del glifosato. El objetivo general de este trabajo fue evaluar los efectos fisiológicos desencadenados por el glifosato que llevan a los individuos de las poblaciones sensible (GS) y resistente (GR) a la muerte, centrándolo fundamentalmente en las consecuencias del tratamiento con glifosato sobre la ruta de los AAA y la ruta de los aminoácidos ramificados (BCAA). Además, se aplicarion mezclas de glifosato y el inhibidor de la AHAS imazamox (inhibidor de la ruta de los BCAA) para profundizar en la regulación de ambas rutas biosintéticas de aminoácidos y su posible regulación cruzada, además de para confirmar o rechazar la posibilidad del empleo de las mezclas como una alternativa a la aplicación del glifosato en solitario. Para estas metas, la respuesta al glifosato de una población sensible (GS) y otra resistente (GR) de A. palmeri obtenidas en Carolina del Norte, fueron comparada a los niveles molecular y bioquímico. En este trabajo se ha confirmado la correlación entre la sobreexpresión génica, el contenido proteico y el nivel de actividad de la enzima EPSPS con el nivel de resistencia de la población GR de A. palmeri. Los altos niveles de variación en el número de copias génicas (CNV) de la enzima EPSPS no tuvieron efectos importantes en los parámetros estudiados: la expresión de las rutas de biosíntesis de los AAA y de los BCAA, el contenido en aminoácidos libres y el contenido en carbohidratos fue similar para ambas poblaciones en condiciones de control. Esto sugiere que la CNV de la enzima EPSPS no tuvo un efecto pleiotrópico importante en la fisiología de las plantas resistentes. La acumulación de siquimato producida tras el tratamiento con glifosato en la población GS (pero no en la GR) indicó que la afección producida en la población sensible fue mayor. Los incrementos en los niveles de aminoácidos totales libres y aromáticos, menores en la población GR que en la GS, confirmaron que el daño producido en la población sensible fue mayor.
Se estudió la respuesta transcripcional de las poblaciones GS y GR al tratamiento con glifosato, y se detectó una inducción general de la expresión de las enzimas de la ruta de los AAA. Se encontraron incrementos muy grandes en los niveles de tránscritos de la enzima Antranilato sintasa mientras que los niveles de tránscritos de la enzima Corismato mutasa no se incrementaron, lo que sugiere un flujo preferencial de carbono hacia la formación de triptófano en vez de tirosina o fenilalanina tras el punto regulatorio de la formación de corismato en presencia del herbicida. La ausencia de respuesta al tratamiento con glifosato en la expresión génica de la ruta biosintética de los BCAA, y de la ruta de los AAA al imazamox, sugirió que no existe regulación cruzada entre las rutas biosintéticas de los AAA y de los BCAA a nivel transcripcional, a pesar de su estrecha relación. Por último, las interacciones entre las mezclas de dos dosis diferentes de glifosato y una de imazamox se estudiaron en A. palmeri. Se detectó un efecto antagonista general en los principales parámetros fisiológicos: siquimato, aminoácidos y niveles de carbohidratos, porque los efectos detectados con las mezclas fueron en su mayoría menores que la adición de los efectos individuales de cada herbicida. Este antagonismo fisiológico general sugiere que al aplicar la mezcla de glifosato e imazamox en campo, las dosis recomendadas no pueden ser disminuidas. Resumiendo, esta tesis describe nuevos aspectos fisiológicos en la caracterización de la población de Carolina del Norte resistente al glifosato y desentraña el modo de acción del glifosato en plantas sensibles y resistentes cuando éste es aplicado individualmente o combinado con imazamox., Over the last century, the production of crops worldwide has substantially increased due to the improvement in crop varieties and in management techniques. One of the most powerful tools used to achieve this increase was herbicides, which eliminates the weeds that compete with the crops for natural resources. However, the sustained use of herbicides has triggered the appearance of resistance in weeds, which is a global agricultural problem that has been exponentially increasing over the last 30 years.
Glyphosate is the most highly used herbicide worldwide due to its effectiveness and due to genetically modified crops that are resistant to this herbicide. The target site of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme in the biosynthetic pathway of the aromatic amino acids (AAAs). One of the worst weeds reported to have resistant populations to glyphosate is Amaranthus palmeri, whose resistance mechanism involves EPSPS gene amplification, which leads to an overexpression of the enzyme target of glyphosate.
The general objective of this work was to evaluate the physiological effects triggered by glyphosate that resulted in plant mortality in sensitive (GS) and resistant (GR) populations, focusing mainly on the consequences of glyphosate treatment in the AAA pathway and the branched-chain amino acid (BCAA) pathway. In addition, mixtures of glyphosate and the AHAS-inhibitor imazamox (an inhibitor of the BCAA pathway) were applied to obtain new insights into the regulation of both amino acid biosynthetic pathways and the possible cross-regulations and to confirm or reject the possibility of the employment of their mixture as an alternative to the application of glyphosate alone. To this end, the response to glyphosate of one glyphosate-sensitive (GS) and one glyphosate-resistant (GR) population of A. palmeri from North Carolina were compared at the molecular and biochemical levels.
In this work, a correlation between EPSPS overexpression, EPSPS protein content and EPSPS activity and the level of resistance of the A. palmeri GR population was confirmed. A high copy number variation (CNV) of EPSPS had no major pleiotropic effect on the studied parameters; the AAA and BCAA pathway expression, free amino acid profiles and carbohydrate contents were similar for both populations in the untreated plants. This suggests that the CNV of EPSPS had no major pleiotropic effects on the physiology of the resistant plants.
The significant increase in shikimate content with increasing doses of glyphosate in the GS population (but not in the GR population) suggests that the damage produced in the sensitive population was higher. The lower general increase in total free and aromatic amino acid (AAA) content in the GR population than in the GS population confirmed that the GS population damage was higher.
The transcriptional response of the GS and GR populations to glyphosate treatment was studied, and a general induction of AAA pathway enzyme expression was identified. Very high increases in the Anthranilate synthase enzyme transcript levels were measured, while Chorismate mutase transcripts were not increased, suggesting a preferential flux of carbon towards the formation of tryptophan instead of tyrosine or phenylalanine after the regulatory point of chorismate formation. The absence of a response of BCAA gene expression to glyphosate treatment and the AAA gene expression after imazamox treatment suggests that no cross regulation exists between the AAA and BCAA pathways at the transcriptional level, despite their close relationship.
Finally, interactions between mixtures of two different doses of glyphosate and one of imazamox were tested on A. palmeri. A general antagonistic effect was detected in the main physiological markers (shikimate, amino acid and carbohydrate levels) because effects that were detected after the application of the mixtures were mostly lower than the effects seen when applying the individual compounds. This general physiological antagonism suggests that the doses cannot be lowered in the mixtures of glyphosate and imazamox to be applied in the field. To summarize, this study describes new physiological insights into the characterization of the glyphosate-resistant weed population of North Carolina and unravels the mode of action of glyphosate on sensitive and resistant plants when applied alone or in combination with imazamox., This work was financially supported by two grants from the Ministerio Español de Economía y Competitividad (AGL-2013-40567R) and (AGL-2016-77531R). Manuel Fernández Escalada has been holder of a fellowship from the Public University of Navarre and he has received the mobility grant from the Public University of Navarre (2016) and the mobility grant from EDONA-La Caixa (2016)., Programa Oficial de Doctorado en Agrobiología Ambiental (RD 1393/2007), Ingurumen Agrobiologiako Doktoretza Programa Ofiziala (ED 1393/2007)

Datos de investigación alojados en: https://doi.org/10.5281/zenodo.16083355, 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., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R) and Spanish Ministry of Science and Innovation (2020 117723-RB-100). Maria Barco-Antoñanzas received funding from PhD fellowship and mobility grant through Universidad Pública de Navarra. Mikel V. Eceiza is the holder of a predoctoral fellowship of the Basque Government. We thank Gustavo Garijo and Clara Jimenez for technical assistance. Open access funding provided by Universidad Pública de Navarra.

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., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R). A.Z.-G and M.B-A. received funding from fellowships through Universidad Pública de Navarra. M.V.E. is the holder of a predoctoral fellowship of the Basque Government., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R). A.Z.-G and M.B-A. received funding from fellowships through Universidad Pública de Navarra. M.V.E. is the holder of a predoctoral fellowship of the Basque Government.

Datos de investigación alojados en: https://doi.org/10.5281/zenodo.16090067, 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., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R), the Public University of Navarre (Project UPNA20-6138) and the Spanish Ministry of Science and Innovation (2020-117723RB-100). M.V. Eceiza is the holder of a predoctoral fellowship of the Basque Government. Open access funding provided by Universidad Pública de Navarra.

Este trabajo se plantea en el marco de la necesidad de profundizar en el conocimiento de la fisiología de las plantas resistentes a herbicidas, lo que requiere, no solo identificar y dilucidar las bases moleculares de las resistencias, sino también abordar el plano fisiológico, analizando la respuesta a nivel de metabolismo a la aplicación de estos herbicidas. La utilización de plantas que ya han desarrollado resistencia múltiple a estos herbicidas es una buena herramienta para desarrollar métodos para su detección, aportar claves para su manejo y prevenir su aparición. Además, el establecimiento de la acción herbicida en plantas resistentes permite comprender cómo y por qué son letales estos herbicidas (conocimiento del modo de acción herbicida), ayudando a que su utilización sea más racional y sostenible.
Se ha profundizado en la fisiología de una población de A. palmeri con resistencia múltiple (RM) a los herbicidas glifosato y piritiobac (inhibidor de ALS) y en los efectos fisiológicos de estos herbicidas. Para ello, se han caracterizado a nivel molecular los mecanismos de resistencia, y se ha abordado la acción herbicida estudiando la respuesta fisiológica a diferentes niveles tras el tratamiento con glifosato, piritiobac o la mezcla de ambos.
Este trabajo proporciona nuevos detalles acerca de la caracterización y de la fisiología de la población RM y aporta información novedosa en cuanto a los efectos fisiológicos provocados en las plantas sensibles y resistentes por los herbicidas IBAR e IBAA. Estos resultados son, potencialmente, de interés práctico para el sector agrícola, por la importancia global de las malas hierbas resistentes a glifosato, herbicida más usado a nivel mundial y a inhibidores de ALS, grupo de herbicidas que incluye un gran número de materias activas. Todo ello, unido al incesante incremento de resistencias múltiples a ambos tipos de herbicidas, hace necesarios los estudios que profundicen en el conocimiento fisiológico de este tipo de resistencias para avanzar hacia un manejo más sostenible y duradero., This work is performed within the framework of the need of obtaining new insights about physiology of resistant weeds. This involves not only identifying and elucidating the molecular bases of resistance, but also addressing the plant physiology, by analyzing the metabolic response to the herbicide application. The study of plants that have already developed multiple resistance to herbicides is a good tool to develop methods to detect them, improve their management and prevent their evolution. Furthermore, the full knowledge of the herbicidal action on resistant plants will unravel how and why these herbicides are lethal (knowledge of the mode of action of the herbicide), helping to make their use more rational and sustainable.
In this study, a full study of the physiology of an A. palmeri population with multiple resistance (RM) to the herbicides glyphosate and pyrithiobac (ALS inhibitor), and of the physiological effects of these herbicides have been approached. For this purpose, the resistance molecular basis has been deciphered and the physiological response to different levels of each herbicide (and their mixture) has been approached to study the herbicide action.
In summary, this work provides new insights on the characterization and physiology of the RM population and unravels the physiological effects of glyphosate and ALS inhibitors on sensitive and resistant plants. These results are, potentially, of great practical and agricultural interest, due to the global significance of weed populations resistant to glyphosate (the most used herbicide worldwide) or to ALS-inhibitors (where a great number of active ingredients are included). All this, together with the continuous evolution of RM populations to both types of herbicides, makes essential to deepen our physiological knowledge of this type of resistances, moving forward a more sustainable and lasting weed management., Ministerio de Economía y Competitividad de España (AGL2016-77531-R). Ayuda predoctoral de la Universidad Pública de Navarra (2017-2021). Dos becas de movilidad, una correspondiente al programa Erasmus+ (2018) y otra procedente de la Escuela de Doctorado de Navarra (EDONA) (2019)., Programa de Doctorado en Agrobiología Ambiental (RD 99/2011), Ingurumen Agrobiologiako Doktoretza Programa (ED 99/2011)

Datos de investigación alojados en: https://doi.org/10.5281/zenodo.16091876, 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., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016- 77531-R) and Spanish Ministry of Science and Innovation (2020 117723-RB-I00).

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., Glyphosate is the most widely used herbicide worldwide. The site of action of this
herbicide is the inhibition of the 5‐enolpyruvylshikimate‐3‐phosphate synthase
(EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway, also
known as shikimate pathway. One of the physiological processes triggered by the
herbicide is quinate accumulation. Quinate is a secondary metabolite, formed in a
lateral branch of the shikimate pathway. The detection of some physiological
effects after applying quinate exogenously raised the possibility of using this
compound in combination with glyphosate to enhance herbicide efficacy and to
reduce the glyphosate doses in the control of Amaranthus palmeri. Glyphosate
resistant populations have evolved in this species, and one of the most important
resistance mechanism is EPSPS gene amplification, which leads to an
overexpression of the enzyme target of glyphosate.
The general objective of this work was to gain further insights in the regulation of
the shikimate pathway after glyphosate treatment and the use of quinate as an
enhancer of this herbicide in the control of glyphosate‐sensitive and resistant
(due to gene amplification) A. palmeri plants.
In this work, it was determined that applying quinate one day after glyphosate
(combined treatment), become lethal a sub lethal glyphosate dose in the
glyphosate‐sensitive population, laying the framework for the application of the
both compounds to improve the efficacy of the herbicide and to reduce the doses
in the control of the sensitive population. The higher toxicity of the combined
treatment was related to an exacerbation of changes in the herbicide
physiological markers previously reported (shikimate and free amino acid
content).
The pattern of the shikimate pathway after the combined treatment was
approached at metabolic and transcriptional level, trying to explain the increase
in the efficacy detected of the combined treatment in this population. The results
obtained suggest that the enhancement of the toxicity observed after the
combined treatment would be related mainly at metabolic level, due to the
increase in the quinate derivative content, and not to changes at transcriptional
level.
A non‐targeted metabolic profiling was performed trying to compare the
metabolic profile of both populations treated with glyphosate and to unravel if
any change in the metabolomic profile of the plants treated with the combined
treatment could contribute to the toxicity increase. Among all primary and secondary metabolites evaluated, the quinate derivatives were the only
compounds that were accumulated differently after the combined treatment,
evidencing their role in the increased toxicity of the combined treatment.
Finally, the role of aromatic amino acids and the intermediates (shikimate,
quinate, chorismate and anthranilate) in the regulation of shikimate pathway was
evaluated by leaf disc incubation. It was observed that aromatic amino acids
when applied combined with the herbicide abolished the glyphosate effects at
transcriptional level but no shikimate accumulation. Also, none of the
intermediates evaluated fully mimicked glyphosate effect on shikimate pathway.
However, shikimate was the metabolite that induced the relative transcript level
of most of the genes of the shikimate pathway, suggesting that the transcription
induction detected after glyphosate treatment would be mediated, at least in
part, by shikimate accumulation.
In summary, this study describes new insights in the shikimate pathway
regulation after glyphosate treatment and evaluates the combined treatment
with quinate from a management and physiological points of view, trying to
unravel the causes of its increase of the toxicity., Ministerio Español de Economía y Competitividad (AGL‐2013‐40567R) and (AGL‐2016‐77531R); Fellowship from the Public University of Navarre; Mobility grants from Gobierno de Navarra (2018), Universidad Pública de Navarra (2018) and Erasmus+ (2018)., Programa de Doctorado en Agrobiología Ambiental (RD 99/2011), Ingurumen Agrobiologiako Doktoretza Programa (ED 99/2011)

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., This work was supported by a grant from the Ministerio Español de Economía y Competitividad (Project number AGL2016-77531-R). M.F.-E. and A.Z.-G. received funding from fellowships trough Universidad Pública de Navarra.

Datos de investigación alojados en: https://doi.org/10.5281/zenodo.15875028, 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., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R), Spain and by the Universidad Pública de Navarra, Spain (Project UPNA20-6138). Although the information is not included in the article, the Spanish Ministry of Science and Innovation was also funded (PID2020-117723RB-100).

Glyphosate is a widely used herbicide targeting the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) in the aromatic amino acid biosynthesis pathway (shikimate pathway) and provoking
accumulation of quinate, a secondary metabolite synthesized through a side branch of this pathway. The objective of this work was to evaluate whether the efficacy of glyphosate activity in Amaranthus palmeri is
enhanced by quinate application one day after herbicide treatment. To this end, one glyphosate-sensitive and one glyphosate-resistant (due to EPSPS gene amplification) population of A. palmeri were used. The 3-
day time course study of the quinate treatment alone showed quinate, Tyr and Phe accumulation in both populations. When the herbicide was applied alone at 0.25× the recommended dose, no phytotoxicity or
glyphosate effects were detected in the sensitive population 3 days after treatment, but the combined treatment with quinate was lethal, and markers of herbicide activity at the amino acid level could be detected.
In the resistant population, an important metabolic perturbation in the flux of the shikimate pathway was detected in the combined treatment. These results raise the possibility of the joint application of quinate and
glyphosate to enhance glyphosate efficacy while lowering doses in the sensitive population., This work was supported by a grant from the Ministerio de Economía y Competitividad , Spain (Project number AGL2016-77531-R ). AZ-G and MF-E received funding from fellowships trough Universidad Pública de Navarra.

The inhibition of acetolactate synthase (ALS; EC 2.2.1.6), an enzyme located in the biosynthetic pathway of branched-chain amino acids, is the target site of the herbicide imazamox. One of the physiological effects triggered after ALS inhibition is the induction of aerobic ethanol fermentation. The objective of this study was to unravel if fermentation induction is related to the toxicity of the herbicide or if it is a plant defense mechanism. Pea plants were exposed to two different times of hypoxia before herbicide application in order to induce the ethanol fermentation pathway, and the physiological response after herbicide application was evaluated at the level of carbohydrates and amino acid profile. The effects of the herbicide on total soluble sugars and starch accumulation, and changes in specific amino acids (branched-chain, amide, and acidic) were attenuated if plants were subjected to hypoxia before herbicide application. These results suggest that fermentation is a plant defense mechanism that decreases the herbicidal effect., This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL2016-77531-R).