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
ERF-VII transcription factors induce ethanol fermentation in response to amino acid biosynthesis-inhibiting herbicides
(Oxford University Press, 2019) Gil Monreal, Miriam; Giuntoli, Beatrice; Zabalza Aznárez, Ana; Licausi, Francesco; Royuela Hernando, Mercedes; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
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.
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, PJUPNA 2010
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
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
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
Cysteine proteases are activated in sensitive Amaranthus palmeri populations upon treatment with herbicides inhibiting amino acid biosynthesis
(Wiley, 2023) Barco Antoñanzas, María; Font Farre, María; Eceiza, Mikel Vicente; Gil Monreal, Miriam; Van der Hoorn, Reiner; 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 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.
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
Role of glutathione S-transferases in the mode of action of herbicides that inhibit amino acid synthesis in Amaranthus palmeri
(Elsevier, 2024) Eceiza, Mikel Vicente; Jiménez Martínez, Clara; Gil Monreal, Miriam; Barco Antoñanzas, María; Font Farre, María; Huybrechts, Michiel; Van der Hoorn, Reiner; Cuypers, Ann; Royuela Hernando, Mercedes; Zabalza Aznárez, Ana; Institute for Multidisciplinary Research in Applied Biology - IMAB; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Acetolactate synthase inhibitors (ALS inhibitors) and glyphosate are two classes of herbicides that act by inhibiting an enzyme in the biosynthetic pathway of branched-chain or aromatic amino acids, respectively. Besides amino acid synthesis inhibition, both herbicides trigger similar physiological effects in plants. The main aim of this study was to evaluate the role of glutathione metabolism, with special emphasis on glutathione S-transferases (GSTs), in the mode of action of glyphosate and ALS inhibitors in Amaranthus palmeri. For that purpose, plants belonging to a glyphosate-sensitive (GLS) and a glyphosate-resistant (GLR) population were treated with different doses of glyphosate, and plants belonging to an ALS-inhibitor sensitive (AIS) and an ALS-inhibitor resistant (AIR) population were treated with different doses of the ALS inhibitor nicosulfuron. Glutathione-related contents, GST activity, and related gene expressions (glutamate-cysteine ligase, glutathione reductase, Phi GST and Tau GST) were analysed in leaves. According to the results of the analytical determinations, there were virtually no basal differences between GLS and GLR plants or between AIS and AIR plants. Glutathione synthesis and turnover did not follow a clear pattern in response to herbicides, but GST activity and gene expression (especially Phi GSTs) increased with both herbicides in treated sensitive plants, possibly related to the rocketing H2O2 accumulation. As GSTs offered the clearest results, these were further investigated with a multiple resistant (MR) population, compressing target-site resistance to both glyphosate and the ALS inhibitor pyrithiobac. As in single-resistant plants, measured parameters in the MR population were unaffected by herbicides, meaning that the increase in GST activity and expression occurs due to herbicide interactions with the target enzymes.
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
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