Person: Fernández Escalada, Manuel
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Fernández Escalada
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Manuel
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Ciencias del Medio Natural
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810924
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Publication Open Access Effects of EPSPS Copy Number Variation (CNV) and glyphosate application on the aromatic and branched chain amino acid synthesis pathways in Amaranthus palmeri(Frontiers Media, 2017) Fernández Escalada, Manuel; Zulet González, Ainhoa; Gil Monreal, Miriam; Zabalza Aznárez, Ana; Ravet, Karl; Gaines, Todd; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaA key enzyme of the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), is the known target of the widely used herbicide glyphosate. Glyphosate resistance in Amaranthus palmeri, one of the most troublesome weeds in agriculture, has evolved through increased EPSPS gene copy number. The aim of this work was to study the pleiotropic effects of (i) EPSPS increased transcript abundance due to gene copy number variation (CNV) and of (ii) glyphosate application on the aromatic amino acid (AAA) and branched chain amino acid (BCAA) synthesis pathways. Hydroponically grown glyphosate sensitive (GS) and glyphosate resistant (GR) plants were treated with glyphosate 3 days after treatment. In absence of glyphosate treatment, high EPSPS gene copy number had only a subtle effect on transcriptional regulation of AAA and BCAA pathway genes. In contrast, glyphosate treatment provoked a general accumulation of the transcripts corresponding to genes of the AAA pathway leading to synthesis of chorismate in both GS and GR. After chorismate, anthranilate synthase transcript abundance was higher while chorismate mutase transcription showed a small decrease in GR and remained stable in GS, suggesting a regulatory branch point in the pathway that favors synthesis toward tryptophan over phenylalanine and tyrosine after glyphosate treatment. This was confirmed by studying enzyme activities in vitro and amino acid analysis. Importantly, this upregulation was glyphosate dose dependent and was observed similarly in both GS and GR populations. Glyphosate treatment also had a slight effect on the expression of BCAA genes but no general effect on the pathway could be observed. Taken together, our observations suggest that the high CNV of EPSPS in A. palmeri GR populations has no major pleiotropic effect on the expression of AAA biosynthetic genes, even in response to glyphosate treatment. This finding supports the idea that the fitness cost associated with EPSPS CNV in A. palmeri may be limited.Publication Open Access The pattern of shikimate pathway and phenylpropanoids after inhibition by glyphosate or quinate feeding in pea roots(Elsevier, 2017) Zabalza Aznárez, Ana; Orcaray Echeverría, Luis; Fernández Escalada, Manuel; Zulet González, Ainhoa; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaThe shikimate pathway is a metabolic route for the biosynthesis of aromatic amino acids (AAAs) (i.e. phenylalanine, tyrosine, and tryptophan). A key enzyme of shikimate pathway (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS) is the target of the widely used herbicide glyphosate. Quinate is a compound synthesized in plants through a side branch of the shikimate pathway. Glyphosate provokes quinate accumulation and exogenous quinate application to plants shows a potential role of quinate in the toxicity of the herbicide glyphosate. Based on this, we hypothesized that the role of quinate accumulation in the toxicity of the glyphosate would be mediated by a deregulation of the shikimate pathway. In this study the effect of the glyphosate and of the exogenous quinate was evaluated in roots of pea plants by analyzing the time course of a full metabolic map of several metabolites of shikimate and phenylpropanoid pathways. Glyphosate application induced an increase of the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS, first enzyme of the shikimate pathway) protein and accumulation of metabolites upstream of the enzyme EPSPS. No common effects on the metabolites and regulation of shikimate pathway were detected between quinate and glyphosate treatments, supporting that the importance of quinate in the mode of action of glyphosate is not mediated by a common alteration of the regulation of the shikimate pathway. Contrary to glyphosate, the exogenous quinate supplied was probably incorporated into the main trunk from the branch pathway and accumulated in the final products, such as lignin, concomitant with a decrease in the amount of DAHPS protein.Publication Open Access Enhancement of glyphosate efficacy on Amaranthus palmeri by exogenous quinate application(Elsevier, 2019) Zulet González, Ainhoa; Fernández Escalada, Manuel; Zabalza Aznárez, Ana; Royuela Hernando, Mercedes; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaGlyphosate 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.Publication 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 PublikoaEn 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.