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
Induction of the PDH bypass and upregulation of the ALDH7B4 in plants treated with herbicides inhibiting amino acid biosynthesis
(Elsevier, 2017) Gil Monreal, Miriam; Zabalza Aznárez, Ana; Missihoun, Tagnon D.; Dormann, Peter; Bartels, Dorothea; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Imazamox and glyphosate represent two classes of herbicides that inhibit the activity of acetohydroxyacid synthase in the branched-chain amino acid biosynthesis pathway and the activity of 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthesis pathway, respectively. However, it is still unclear how imazamox and glyphosate lead to plant death. Both herbicides inhibit amino-acid biosynthesis and were found to induce ethanol fermentation in plants, but an Arabidopsis mutant deficient in alcohol dehydrogenase 1 was neither more susceptible nor more resistant than the wild-type to the herbicides. In this study, we investigated the effects of the amino acid biosynthesis inhibitors, imazamox and glyphosate, on the pyruvate dehydrogenase bypass reaction and fatty acid metabolism in A. thaliana. We found that the pyruvate dehydrogenase bypass was upregulated following the treatment by the two herbicides. Our results suggest that the Arabidopsis aldehyde dehydrogenase 7B4 gene might be participating in the pyruvate dehydrogenase bypass reaction. We evaluated the potential role of the aldehyde dehydrogenase 7B4 upon herbicide treatment in the plant defence mechanism. Plants that overexpressed the ALDH7B4 gene accumulated less soluble sugars, starch, and fatty acids and grew better than the wild-type after herbicide treatment. We discuss how the upregulation of the ALDH7B4 alleviates the effects of the herbicides, potentially through the detoxification of the metabolites produced in the pyruvate dehydrogenase bypass.
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
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 Publikoa
A 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.
Open Access
Proteolytic pathways induced by herbicides that inhibit amino acid biosynthesis
(Public Library of Science, 2013) Zulet González, Amaia; Gil Monreal, Miriam; Villamor, Joji Grace; Zabalza Aznárez, Ana; Hoorn, Renier A.L. van der; Royuela Hernando, Mercedes; Ciencias del Medio Natural; Natura Ingurunearen Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Background: The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results: Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides.Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion: These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined
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
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.