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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). M.B.-A. received funding from fellowship through Universidad Pública de Navarra. M.V.E. is the holder of a predoctoral fellowship of the Basque Government.

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.

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&ndash, 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.

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., ERF-VII transcription factors regulate the induction of ethanol fermentation upon amino acid biosynthesis-inhibiting herbicide treatment in plants grown with no oxygen limitations. A reduced fermentative potential enhances sensitivity to herbicides.

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 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.

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.

1ª edición Enlace a la versión en euskera: https://hdl.handle.net/2454/39451 El origen de este libro se encuentra en el sitio web de Flora Arvense de Navarra, que fue desarrollado con el objetivo de facilitar el conocimiento de las malas hierbas de Navarra al alumnado de la Escuela Técnica Superior de Ingenieros Agrónomos de la Universidad Pública de Navarra, además de el de ser útil para personal investigador, técnico y agricultores de Navarra y territorios próximos. La edición de este libro se planteó para permitir la disponibilidad del contenido del sitio web en un soporte independiente de Internet.

1. edizioa Enlace a la versión en castellano: https://hdl.handle.net/2454/39450 Liburu honen jatorria Flora Arvense de Navarra webgunea izan da. Weba ikasleei laguntzeko asmoz sortu eta garatu zen; hain zuzen ere, Nafarroako Unibertsitate Publikoko Nekazaritza Ingeniarien Goi Mailako Eskola Teknikoko ikasleei Nafarroako belar gaiztoen gaineko ezagutza errazteko. Aldi berean, erabilgarria da Nafarroako eta inguruko ikertzaile, teknikari eta nekazarientzat.

Incluye 2 ficheros de datos The herbicide glyphosate inhibits the plant enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS) in the aromatic amino acid (AAA) biosynthetic pathway. The physiologies of an Amaranthus palmeri population exhibiting resistance to glyphosate by EPSPS gene amplification (NC-R) and a susceptible population (NC-S) were compared. The EPSPS copy number of NC-R plants was 47.5-fold the copy number of NC-S plants. Although the amounts of EPSPS protein and activity were higher in NC-R plants than in NC-S plants, the AAA concentrations were similar. The increases in total free amino acid and in AAA contents induced by glyphosate were more evident in NC-S plants. In both populations, the EPSPS protein increased after glyphosate exposure, suggesting regulation of gene expression. EPSPS activity seems tightly controlled in vivo. Carbohydrate accumulation and a slight induction of ethanol fermentation were detected in both populations. This work was financially supported by a grant from the Ministerio Español de Economıa y Competitividad (AGL- 2013-40567R). M.F.-E. and M.G.-M. received funding from fellowships through the Universidad Publica de Navarra.

Incluye 3 ficheros de datos 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. Miriam Gil-Monreal received funding from fellowships through Universidad Pública de Navarra. This work was financially supported by two grants from the Ministerio Español de Economía y Competitividad (AGL-2013-40567R and AGL-2016-77531R).

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. MF-E, AZ-G, and MG-M received funding from fellowships trough Universidad Pública de Navarra. This work was financially supported by a grant from the Ministerio Español de Economía y Competitividad (AGL-2016-77531R). This work was also partially financially supported by the USDA National Institute of Food and Agriculture, Hatch project COL00719 to the Colorado State University Agricultural Experiment Station.

The 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. This work was financially supported by a grant from the Ministerio Español de Economía y Competitividad (AGL-2013-40567R). L.O. received a grant from the Ministerio de Educación. M. F-E and AZ-G received funding from fellowships from Universidad Pública de Navarra.

This is the peer reviewed version of the following article: Armendáriz, O., Gil-Monreal, M., Zulet, A., Zabalza, A. and Royuela, M. (2016), Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots. Plant Biol J, 18: 382–390. doi:10.1111/plb.12412, which has been published in final form at http://dx.doi.org/10.1111/plb.12412. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. 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. A. Zulet and M. Gil-Monreal received funding from fellowships through the Ministerio de Educación and Universidad Pública de Navarra, respectively. This work was financially supported by a grant from the Ministerio Español de Economía y Competitividad (AGL-2013-4067R).

Plant internal oxygen concentrations can drop well below ambient even when the plant grows under optimal conditions. Using pea (Pisum sativum) roots, we show how amenable respiration adapts to hypoxia to save oxygen when the oxygen availability decreases. The data cannot simply be explained by oxygen being limiting as substrate but indicate the existence of a regulatory mechanism, because the oxygen concentration at which the adaptive response is initiated is independent of the actual respiratory rate. Two phases can be discerned during the adaptive reaction: an initial linear decline of respiration is followed by a nonlinear inhibition in which the respiratory rate decreased progressively faster upon decreasing oxygen availability. In contrast to the cytochrome c pathway, the inhibition of the alternative oxidase pathway shows only the linear component of the adaptive response. Feeding pyruvate to the roots led to an increase of the oxygen consumption rate, which ultimately led to anoxia. The importance of balancing the in vivo pyruvate availability in the tissue was further investigated. Using various alcohol dehydrogenase knockout lines of Arabidopsis (Arabidopsis thaliana), it was shown that even under aerobic conditions, alcohol fermentation plays an important role in the control of the level of pyruvate in the tissue. Interestingly, alcohol fermentation appeared to be primarily induced by a drop in the energy status of the tissue rather than by a low oxygen concentration, indicating that sensing the energy status is an important component of optimizing plant metabolism to changes in the oxygen availability.

[Background] The increased selection pressure of the herbicide glyphosate has played a role in the evolution of glyphosate-resistance in weedy species, an issue that is becoming a threat to global agriculture. The molecular components involved in the cellular toxicity response to this herbicide at the expression level are still unidentified. [Results] In this study, we identify the protein kinase GCN2 as a cellular component that fosters the action of glyphosate in the model plant Arabidopsis thaliana. Comparative studies using wild-type and gcn2 knock-out mutant seedlings show that the molecular programme that the plant deploys after the treatment with the herbicide, is compromised in gcn2. Moreover, gcn2 adult plants show a lower inhibition of photosynthesis, and both seedlings and adult gcn2 plants accumulate less shikimic acid than wild-type after treatment with glyphosate. [Conclusions] These results points to an unknown GCN2-dependent factor involved in the cascade of events triggered by glyphosate in plants. Data suggest either that the herbicide does not equally reach the target-enzyme in a gcn2 background, or that a decreased flux in the shikimate pathway in a gcn2 plants minimize the impact of enzyme inhibition. © 2015 Faus et al.; licensee BioMed Central., This work was mainly supported by the Universidad Politecnica de Valencia (PAID2011-16) and the Ministerio Español de Ciencia y Tecnología (BFU2011-22526). The work was partially supported through a grant from the Ministerio Español de Ciencia y Tecnología (AGL-2010-18621). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

The adaptation of the respiratory metabolism in roots of soybean (Glycine max L. Merr. cv Ransom) treated with herbicides that inhibit the enzyme acetolactate synthase (ALS) was analyzed. A new gas phase dual-inlet mass spectrometry system for simultaneous measurement of 34O2 to 32O2 and O2 to N2 ratios has been developed. This system is more accurate than previously described systems, allows measurements of much smaller oxygen gradients, and, as a consequence, works with tissues that have lower respiration rates. ALS inhibition caused an increase of the alternative oxidase (AOX) protein and an accumulation of pyruvate. The combination of these two effects is likely to induce the activation of the alternative pathway and its participation in the total respiration. Moreover, the start of the alternative pathway activation and the increase of AOX protein were before the decline in the activity of cytochrome pathway. The possible role of AOX under ALS inhibition is discussed.

Incluye 1 fichero de datos 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. This work was financially supported through a grant from the Ministerio Espanol de Ciencia y Tecnología (AGL-2010-18621 and AGL-2013-40567-R). A. Zulet and M. Gil-Monreal received funding from fellowships through the Ministerio de Educación and the Universidad Pública de Navarra, respectively.

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 A. Zulet and M. Gil-Monreal received funding from Spanish fellowships through the Ministerio de Educación and the Universidad Pública de Navarra, respectively. This work was financially supported through grants from the Spanish Ministry of Education and Science (AGL-2010-18621) and Max Planck Society and the COST Action CM1004 for funding.

The final publication is available at Springer via http://dx.doi.org/10.1007/s00344-013-9345-5 Quinate (1,3,4,5-tetrahydroxycyclohexanecarboxylate) is a compound synthesized in plants through a side branch of the shikimate biosynthesis pathway. Plants treated with herbicides that inhibit amino acid biosynthesis (branched-chain and aromatic) accumulate quinate in their leaves. The objective of this study was to evaluate whether quinate mimics the effects of herbicides in plants. In pea plants, exogenous application of quinate through the nutrient solution was compared with leaf spraying at a concentration of 4 and 400 mM, respectively, and evaluated in parallel to the effects of herbicides. The analysis facilitated an assessment of the phytotoxicity and potential use of quinate as a natural herbicide. The application of quinate through the nutrient solution, but not the spray, was lethal, although both treatments affected plant growth. Quinate was absorbed and translocated to other plant organs remote from the application site, and an increase in the levels of aromatic amino acids and caffeic acid (that is, compounds located after quinate in the shikimate biosynthesis pathway) was detected, which indicates that quinate was metabolized and incorporated into the shikimate pathway. Exogenous application of quinate affected the carbohydrate content in the leaves and roots in a way similar to the toxic effects of herbicides. The phytotoxic effects of quinate reported in this study suggest that this compound deregulates the shikimate pathway and mimics some physiological effects described in the mode of action of herbicides inhibiting amino acid biosynthesis. A. Zulet received a grant from the Spanish Ministry of Education and Science. This work was supported through funding from the Spanish Ministry of Science and Innovation (AGL- 2010-18621/AGR).

Imazethapyr is a selective herbicide used to control a wide spectrum of weeds in several legume crops, including pea. The effect of imazethapyr on pea-Rhizobiumsymbiosis was studied through its effect on microorganism growth, microorganism nodulation ability and plant growth. Symbiotic plants were damaged by imazethapyr concentrations higher than 1.73 μM applied preemergence. The number of nodules per plant was affected more than nodule size, suggesting a direct imazethapyr effect on the nodule initiation rather than on nodule development. However, imazethapyr did not directly affectRhizobiumbecause doses higher than 0.34 mM were required to cause slight effects onRhizobiumgrowth in a defined medium. Also, nodulation ability of bacteria treated with imazethapyr was not affected. These results suggest that imazethapyr inhibits the growth of the symbiotic plant rather than having a direct effect on the bacteria. Accordingly, symbiotic plants were less affected than the corresponding nitrate-reducing pea plants.

Acetolactate synthase (ALS; EC 4.1.3.18) and ketol-acid reductoisomerase (KARI; EC 1.1.1.86) are two consecutive enzymes in the biosynthesis of branched-chain amino acids. Several commercial herbicides inhibit ALS as their primary site of action. KARI has also attracted attention as a potential target for herbicides. Although potent and selective inhibitors of KARI have been discovered, these inhibitors display less herbicidal activity than ALS-inhibiting herbicides. To obtain a better understanding of these findings, we have compared the physiological effects induced in pea plants after KARI or ALS inhibition. Although, both types of inhibitors induce growth arrest and photosynthesis inhibition, plant death occurs more rapidly under ALS inhibition than KARI inhibition. Carbohydrates accumulated in the leaves and roots following treatments with both inhibitors. The carbohydrate accumulation in the leaves occurred as a consequence of a decrease in sink strength. In contrast, the free amino acid content was only affected through ALS inhibition. These results indicate that although KARI and ALS inhibition block the same biosynthetic pathway and exert common effects on carbon metabolism, nitrogen metabolism is more affected via ALS than KARI inhibition. Thus, metabolic alterations in nitrogen metabolism induced through ALS inhibitors might contribute to the increased efficacy of these chemicals as herbicides. M. Igal received a grant from the Public University of Navarre. A. Zulet and M. Gil-Monreal receive funding through fellowships from the Spanish Ministry of Education and the Public University of Navarre, respectively. This work was financially supported through grants from the Spanish Ministry of Education and Science (AGL-2010-18621).

The herbicide glyphosate reduces plant growth and causes plant death by inhibiting the biosynthesis of aromatic amino acids. The objective of this work was to determine whether glyphosate-treated plants show a carbon metabolism pattern comparable to that of plants treated with herbicides that inhibit branched-chain amino acid biosynthesis. Glyphosate-treated plants showed impaired carbon metabolism with an accumulation of carbohydrates in the leaves and roots. The growth inhibition detected after glyphosate treatment suggested impaired metabolism that impedes the utilization of available carbohydrates or energy at the expected rate. These effects were common to both types of amino acid biosynthesis inhibitors. Under aerobic conditions, ethanolic fermentative metabolism was enhanced in the roots of glyphosate-treated plants. This fermentative response was not related to changes in the respiratory rate or to a limitation of the energy charge. This response, which was similar for both types of herbicides, might be considered a general response to stress conditions. L. Orcaray and A. Zulet were the recipients of grants from the Spanish Ministry of Education. This work was supported by the Spanish Ministry of Education and Science (AGL-2007-61134/AGR and AGL-2010- 18621).

Chlorsulfuron was preemergence applied in field trials at La Corufia, Spain, at rates from 5 to 30 g ai ha-1 (1987-88) and from 5 to 100 g ai ha71 (1988-89) for weed control in winter and spring wheat. Excellent control of broadleaf weeds was obtained in both; however, narrowleaf control at the last evaluation (heading) was poor. Chlorsulfuron at 30 g ha-1 did not cause a residual problem in 1987-88, with only 0.24 jig kg-1 of dry soil recovered after both a winter and spring wheat cropping season. However, chlorsulfuron persistence at the same rate was very high in the dry year 1988-89, with 0.43 jg kg71 and 0.53 jg kr1 recovered in winter and spring wheat, respectively. 'Cardeno' spring wheat showed no phytotoxic symptom at any rate of chlorsulfuron from 5 to 100 g ha71. Its yield and yield components (spikes m-2, grain number spike-1, grain weight) were greater with all chlorsulfuron rates, but not in 'Fiel' winter wheat for rates of 30 g ha71 or higher. Nomenclature: Chlorsulfuron, 2-chloro-N-(((4-methoxy- 6 - methyl - 1,3,5-triazin - 2 - yl)-amino)carbon- yl)benzenesulfonamide; wheat, Triticum aestivum L. Cardeno and Fiel. Additional index words. Bioassay, efficacy, tolerance.

Nitrogen fixation in legume nodules has been shown to be very sensitive to drought and other environmental constraints. It has been widely assumed that this decline in nitrogen fixation was a consequence of an increase in the so-called oxygen diffusion barrier and a subsequent impairment to bacteroid respiration. However, it has been recently shown that nitrogen fixation is highly correlated with nodule sucrose synthase (SS) activity under drought and other environmental stresses. Whether this correlation reflects a causative relationship or not has not been proven yet. The evidence presented here suggests that SS controls nitrogen fixation under mild drought conditions. However, nitrogen fixation cannot be enhanced only by increasing glycolytic flux, as under these conditions nodules become oxygen limited. Abscisic acid also induces a decline in nitrogen fixation that is independent of SS. The overall results suggest the occurrence of a complex regulation of nodule nitrogen fixation involving, at least, both carbohydrate and oxygen fluxes within the nodule. Loli Gálvez was the holder of a grant from the Spanish Ministry of Education (Plan F.P.U.). This work was supported by DGESIC (PB98-0545).