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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.

Background: Hydroponic systems are a convenient platform for plant cultivation when treatments are applied to the roots because they provide precise control of the composition of the growth medium, ensuring the availability of different compounds. A problem arises when axenic conditions are needed but the treatment of choice (exogenous organic acids or sugars) promote the growth of unwanted microorganisms. Moreover, axenic conditions are usually applied in liquid and semi-liquid growing systems, where oxygen availability can be compromised, if no aeration is provided. Results: The driver for the development of this hydroponic system was the application of the organic acid pyruvate to the roots of plants grown under aerated axenic conditions. No contamination was detected in the nutrient solution, even after the addition of pyruvate. The system was validated in pea plants treated with either pyruvate or herbicides inhibiting amino acid biosynthesis. The effects on ethanol fermentation were compared by analysing the enzymatic activity, protein content and transcriptional levels in plants treated with either pyruvate or herbicides. Conclusions: The developed system enables the study of the exogenous application of organic acids in the nutrient solution under axenic conditions and without oxygen limitation. This system allows the study of the effect of any type of treatments applied to roots under aerated axenic hydroponic systems at physiological and molecular levels. The role of pyruvate in the induction of fermentation by herbicides cannot be simply explained by an increase in substrate availability. MGM and MFE 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-2016-7531R).

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).

Chlorsulfuron and imazethapyr (herbicides that inhibit acetolactate synthase; ALS, EC 4.1.3.18) produced a strong accumulation of hydroxycinnamic acids that was related to the induction of the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 2.5.2.54). The exogenous application of two hydroxycinnamic acids, ferulic and p-coumaric acids, to pea plants resulted in their internal accumulation, arrested growth, carbohydrate and quinate accumulation in the leaves, and the induction of ethanolic fermentation. These effects resemble some of the physiological effects detected after acetolactate synthase inhibition and suggest important roles for ferulic and p-coumaric acids in the mode of action of herbicides inhibiting the biosynthesis of branched chain amino acids.

Herbicides that inhibit branched chain amino acid biosynthesis induce aerobic fermentation. The role of fermentation in the mode of action of these herbicides is not known, nor is the importance of this physiological response in the growth inhibition and the lethality caused by them. Metabolic profiling was used to compare the effects of the herbicide imazethapyr (IM) on pea plants with two other treatments that also induce fermentation: hypoxia and the exogenous supply pyruvate for seven days. While hypoxic roots did not show internal anoxia, feeding pyruvate or applying IM to the roots led to internal anoxia, probably related to the respiratory burst detected. The three treatments induced ethanol fermentation, but fermentation induced following herbicide treatment was earlier than that following pyruvate supply and was not associated with a decrease in the energy status. No striking changes were detected in the metabolic profiling of hypoxic roots, indicating that metabolism was only slightly impaired. Feeding pyruvate resulted in marked succinate accumulation and a general amino acid accumulation. IM-treated roots showed a general accumulation of glycolytic metabolites upstream of pyruvate, a decrease in some TCA intermediates and an increase in the free amino acid pool sizes. All treatments caused GABA and putrescine accumulation. Our results indicate that IM supply impairs carbon/nitrogen metabolism and this impaired metabolism is likely to be related to the growth arrest detected. As growth is arrested, carbohydrates and glycolytic intermediates accumulate and energy becomes more available.

BACKGROUND: The herbicide glyphosate inhibits the biosynthesis of aromatic amino acids by blocking the shikimate pathway. Imazethapyr and chlorsulfuron are two herbicides that act by inhibiting branched-chain amino acid biosynthesis. These herbicides stimulate secondary metabolism derived from the aromatic amino acids. The aim of this study was to test if they cause any cross-effect in the amino acid content and if they have similar effects on the shikimate pathway. RESULTS: The herbicides inhibiting two different amino acid biosynthesis pathways showed a common pattern in general content of free amino acids. There was a general increase in total free amino acid content, with a transient decrease in the proportion of amino acids whose pathways were specifically inhibited. Afterwards, an increase in these inhibited amino acids was detected; this was probably related to proteolysis. All herbicides caused quinate accumulation. Exogenous application of quinate arrested growth, decreased net photosynthesis and stomatal conductance and was ultimately lethal, similarly to glyphosate and imazethapyr. CONCLUSIONS: Quinate accumulation was a common effect of the two different classes of herbicide. Moreover, exogenous quinate application had phytotoxic effects, showing that this plant metabolite can trigger the toxic effects of the herbicides. This ability to mimic the herbicide effects suggests a possible link between the mode of action of these herbicides and the potential role of quinate as a natural herbicide. Copyright © 2009 Society of Chemical Industry

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.

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. -1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.

Imazethapyr (IM) is an imidazolinone herbicide which inhibits the biosynthesis of branched chain amino acids, by blocking acetolactate synthase (ALS; EC 4.1.3.18), the first common enzyme of the pathway. To study new aspects of the mode of action of ALS-inhibiting herbicides, pea plants grown in hydroponic cultures were supplied with IM and were analysed with reference to the antioxidant system and oxidative markers. A slight lipid peroxidation was detected in leaves after IM treatment, but no changes were noted in electrolyte leakage or carbonyl content. The ascorbate pool of leaves was oxidized under IM treatment. The analysis of the antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT) and guaiacol peroxidase (GPX), showed that IM treatment only caused an enhancement of GPX activity in leaves. In roots, the herbicide caused a decrease in lipid peroxidation. The enhancement of the reduced glutathione content detected in IM-treated roots can be related to the detected increase of GR activity. The lack of more noticeable effects on antioxidant enzymatic activities could be explained by the inability of IM-treated plants to respond to oxidative stress with modifications in their protein synthesis. Our results suggest that oxidative stress is not related to the mode of action of ALS-inhibitors. The slight changes detected in the antioxidative status of treated plants are too secondary in time and intensity to be related to the lethality caused by ALS-inhibitors.

[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.

Semi-leafless varieties of pea have considerable agronomic importance and it has been suggested that they may have a superior response to water deficits than conventional varieties. However, these varieties are poorly characterized from a physiological point of view and there is lack of a physiological basis for their supposed better performance under conditions of water deficit. Here, we describe the solute distribution in the different leaf structures of a semi-leafless pea variety (Pisum sativum L.) under non-limiting water conditions and under water stress. A conventional variety was subjected to the same conditions for comparative purposes. A detailed study was carried out both at the tissue level and at the single cell level. In control conditions, epidermal vacuoles of tendrils showed a different ion distribution of those of the laminar leaf structures. However, under water deficit, only stipules of the semi-leafless variety showed a significantly higher capability to increase osmolarity. This occurred by accumulating potassium, magnesium and chloride to a higher extent than other leaf structures. The inability of performing an adequate osmotic adjustment in tendrils may be the cause of the lack of a better response to water deficit.

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.

The recessive gene af produces a modification of Pisum sativum L. leaf morphology, where leaflets are replaced by tendrils. Previous reports have suggested that tendrils may contribute to plant growth in a similar way to flat leaf structures, but these reports have been restricted to carbon metabolism. In this work, we investigate the nitrate reduction (nitrate reductase activity) capacity of tendrils of a semi-leafless variety, Solara, in relation to other leaf structures. Maximum nitrate reductase activity (EC 1.6.6.1), expressed on a protein basis, was significantly lower in tendrils compared to flat structures. However, the activation state of nitrate reductase was significantly higher in tendrils, reaching 70%, compared to flat leaf structures. According to these results, tendrils contributed up to 25% of the overall plant nitrate reduction in the semi-leafless variety. This figure was even higher when nitrate reduction was calculated from in vivo measurements. The results are discussed in relation to nitrate, magnesium, carbohydrates, amino acids and adenylate levels of tendrils.

Acetolactate synthase (ALS) activity extracted from Rhizobium leguminosarum biovar. viciae has been characterized. The optimum pH for extraction was 7.6 and for the assay 7.0. The K m for pyruvate was 7.2 mM, and the enzyme was saturated at 40 mM. An obligatory requirement of TPP and Mg 2+ for full ALS activity was observed. Valine was the only branched-chain amino acid that caused ALS feedback inhibition. The specific activity of Rhizobium ALS was nearly 20 times the activity found in pea (Pisum sativum) leaves. Bacteroids from pea nodules also showed high ALS activity, and the nodule plant fraction had higher ALS activity than other plant tissues. ALS sensitivity to imazethapyr was also dependent on the source: ALS activity of free-living Rhizobium and bacteroids was slightly more tolerant than that of other pea tissues, but the differences were less than those found in rates of specific activity. It is proposed that the high ALS activity expressed by Rhizobium, both as free-living bacteria and as bacteroids, is related to the growth tolerance of rhizobia to imazethapyr and is also related to the relative tolerance of symbiotic pea plants.

Nowadays, there are two main types of herbicides that inhibit amino acid biosynthesis: glyphosate, which inhibits aromatic amino acid synthesis via specific inhibition of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS), and inhibitors of branched-chain amino acid synthesis that act via specific inhibition of acetolactate synthase (ALS). Both types of inhibitors share different aspects of their mode of action, such as the induction of fermentation. Although this physiological effect resembles the hypoxic stress response, it was detected under aerobic conditions and it was not related to a change in respiratory rates or to a decrease in the energy charge. Fermentative induction has also been detected after treatment with other compounds inhibiting amino acid biosynthesis, such as glufosinate or ketol-acid reductoisomerase inhibitors suggesting that it can be considered as part of a general plant response to the stress conditions caused by this kind of herbicide treatment. Fermentation seems to be part of the impaired carbon metabolism detected in these treated plants that prevent the optimal utilization of available carbohydrates and energy.

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).

Oxygen shortage in soils can occur following a wide range of natural circumstances, affecting the plant's physiology. In this paper the performance of nodulated lucerne plants under severe hypoxia is examined and the mechanisms involved to achieve this adaptation are discussed. Nodulated lucerne plants (Medicago sativa L.) were grown with their rooting medium exposed to 1 or 21 kPa oxygen. Final yield, as expressed on a shoot dry weight basis, was unaffected but root and nodule dry weights were reduced by 50%. Water content in roots and nodules was higher at 1 kPa as a result of the formation of aerenchyma. Specific acetylene reduction activity was higher in hypoxic nodules as a consequence of modified nodule structure, although they were more sensitive to the presence of acetylene or nitrate. Root respiration was insensitive to changes in external oxygen supply, therefore providing adequate support for mineral uptake. Nodule respiration rates were 5 times higher in control plants when measured as CO2 evolution, whereas no differences were observed in O2 uptake. It is suggested that adaptation of nodulated lucerne to low oxygen concentrations involves changes in photosynthate allocation and nodule morphology, which provide a more efficient nitrogen fixation.

Summary Maize plants fed with PPT (a glutamine synthetase inhibitor) accumulate ammonia. Approximately 50 % of the ammonia accumulated seems to come from the photorespiratory pathway, while the nonphotorespiratory ammonia is derived from nitrate reductase activity. The ammonia accumulated, however, is not sufficient to increase GDH activity. We also carried out a comparative study of the effect of PPT and AOA on photosynthesis. Both compounds inhibit the photorespiratory pathway, causing glycolate accumulation and diminishing photosynthesis.

Nodulated lucerne plants (Medicago sativa L. cv. Aragon) were supplied with 20 mM nitrate. Anaerobically isolated bacteroids of Rhizobium meliloti from these plants were able to denitrify after 48 h treatment. R. meliloti bacteroids behave as total denitrifiers, reducing nitrate to dinitrogen: when acetylene was omitted from the assay medium very little nitrous oxide was recovered. The onset of denitrification activity was coincident with the induction of nitrite reductase activity (EC 1.7.99.3) whereas nitrate reductase activity (EC 1.7.99.4) was constitutive. Whole nodules from plants receiving several doses of nitrate were assayed, in a nitrate-free medium, to monitor denitrification activity dependent on nitrate within the nodules. Denitrification activity was detected after 2 days of 20 mM nitrate supply or after 3 days in the presence of 10 or 5 mM nitrate. These results are discussed in relation to current controversy about nitrate entry into the infection region of nodules. It is concluded that this process occurs more rapidly than suggested in recent research.

Summary Differences in sensitivity to chlorsulfuron from 100 up to 1000-fold were observed between wheat and maize plants. Free amino acid content was analyzed in wheat and maize embryos and plants after 24 h and 10 days of chlorsulfuron treatment, respectively. Accumulation of free amino acids and increase in relative proportion of some of them were observed even when decreases in branched amino acid content could hardly be measured. Acetolactate synthase extracted from chlorsulfuron treated plants showed the same levels of extractable enzyme as from untreated plants. An effect of chlorsulfuron on acetolactate synthase in vivo could not be detected in wheat or maize at any time of plant treatment; this strongly suggests that the enzyme-inhibitor complex does not remain bound throughout the enzyme extraction procedure. Moreover, the extractable enzyme had the same properties as acetolactate synthase of untreated plants, showing that the enzyme in vivo is not irreversibly damaged by chlorsulfuron. This result is further supported by the growth recovery found in treated plants after removing the herbicide.

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.

The inhibition of branched-chain amino acid (BCAA) biosynthesis was evaluated in pea plants in relation to the ability for induction of fermentative metabolism under aerobic conditions. Chlorsulfuron and imazethapyr (inhibitors of acetolactate synthase, ALS, EC 4.1.3.18) produced a strong induction of pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) activities and a lesser induction of lactate dehydrogenase (LDH, EC 1.1.1.27) and alanine aminotransferase (AlaAT, EC 2.6.1.2) activities in roots. Inhibition of the second enzyme of the BCAA biosynthesis (ketol-acid reductoisomerase, KARI, EC 1.1.1.86) by Hoe 704 (2-dimethylphosphinoyl-2-hydroxyacetic acid) and CPCA (1,1-cyclopropanedicarboxylic acid) enhanced fermentative enzyme activities including PDC, ADH, and AlaAT. Fermentative metabolism induction occurring with ALS- and KARI-inhibitors was related to a higher expression of PDC. In the case of KARI inhibition, it is proposed that fermentation induction is due to an inhibition of ALS activity resulted from an increase in acetolactate concentration. Fermentative metabolism induction in roots, or at least ethanolic fermentation, appeared to be a general physiological response to the BCAA biosynthesis inhibition.

Herbicides that inhibit branched chain amino acid biosynthesis produce a rapid carbohydrate increase in leaves of treated plants. The relationship between these processes is not known nor is the importance of carbohydrate accumulation in the growth inhibition caused by these herbicides. This work analyzes carbohydrate concentration in sources and sinks after herbicide treatments in pea (Pisum sativum L.), as well as photosynthetic carbon assimilation, using two classes of chemicals, chlorsulfuron and imazethapyr, applied to roots or leaves. The most remarkable result was that, in addition to carbohydrate accumulation in leaves, accumulation of sucrose and/or starch in roots was detected. This pattern of carbohydrate accumulation was similar for both herbicides and independent of whether the herbicides were applied to leaves or roots. This indicates that root growth inhibition was not caused by sugar starvation in sinks. Nevertheless, the results are consistent with a decrease in sink strength, leading to the inhibition of photoassimilate translocation.

Acetolactate synthase (ALS; EC 4.1.3.18) inhibition is the primary mechanism of action of imazethapyr (IM). However, the precise mechanisms that links ALS inhibition with plant death have not been elucidated. Supply of IM to pea (Pisum sativum L) plants produced an immediate cessation of growth, caused a 50% inhibition of the in vivo ALS activity within 1 day of treatment, and a remarkable accumulation (2.7-times) of free amino acids after 3 days. Carbohydrates (soluble and starch) were accumulated in both leaves and roots. Accumulation of soluble sugars in roots preceded that of starch in leaves, suggesting that the accumulation of carbohydrates in leaves is not the reason for the arrested root growth. A transient pyruvate accumulation was observed in roots, 1 day after the onset of IM supply. This was coincident with an increase in pyruvate decarboxylase (EC 4.1.1.1), and later increases in alcohol dehydrogenase (EC 1.1.1.1), lactate dehydrogenase (EC 1.1.1.27), and alanine amino transferase (EC 2.6.1.2) activities. This enhancement of fermentative activities was coincident with a slight decrease in aerobic respiration. The overall data suggest that the impairment of ALS activity may lead to a fermentative metabolism that may be involved in growth inhibition and plant death.

It has been suggested that semileafless varieties of pea (Pisum sativum L.), where leaflets are replaced by tendrils, are more tolerant to water deficit than conventional leafed varieties. This response to water stress has been assumed to be related to the reduced leaf area of the semileafless varieties. However, total leaf area of the varieties used in this study was not significantly different and, also, transpiration rate per plant did not differ under control conditions. Moreover, transpiration rates declined similarly in both varieties under water stress. In order to gain further insights into the better tolerance of semileafless plants to water stress, a detailed study was carried out both at tissue level and at single cell level in order to characterise the osmotic adjustment carried out in the different leaf structures. At tissue level, osmolarity did not differ among structures, and it increased similarly under water stress regardless of the leaf structure. While, at mesophyll vacuole level, most mineral elements analysed did not accumulate in response to water stress. At epidermal vacuole level, tendrils of the semileafless showed a higher osmolarity than the homologous structure of conventional pea. This might represent an advantage in order to face water restrictions. Since tendrils represent around a 40% of the compound leaf of the semileafless variety, a more efficient osmotic adjustment carried out by tendrils may represent an important improvement in the water use efficiency under water deficits.

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).

When nitrogen fixing legumes are subjected to drought, it is not obvious which function of the stressed plant is actually affecting the nodule. There is a direct correlation between the decreased nodule water potential and the decline of nitrogenase activity (Pankhurst, Sprent, 1975). Recently, the idea that metabolic potential of nodules is reduced during drought has been confirmed (Diaz del Castillo et al., 1994). We have suggested a key role for sucrose synthase in nodules of soybean subjected to drought (Gonzalez et al., 1995). Our results suggest that this observation can be also assumed for temperate legumes with indeterminate nodules.

Summary Chlorella pyrenoidosa Pringsheim (211/8 a) was grown photoautotrophically using the herbicide, glyphosate [N-(phosphonomethyl)-gycine], in concentrations ranging from 0.1 mM to 1 mM. Chlorophyll and carotenoid content, greening process, photosynthetic and respiration rates, and photosynthetic electron transport activities were investigated. Glyphosate decreased cell density and photosynthetic pigment content; 1.0 mM did not allow growth. Bleached cells were used for the greening process. Glyphosate had two different effects upon photosynthetic pigments: inhibition of chlorophyll synthesis and a decrease in carotenoids. Oxygen uptake was not affected, but oxygen evolution was strongly inhibited. The results suggest that glyphosate acts as an electron transport inhibitor, acting on both photosystems, but its effect was greater on PS II than PS I.

Summary The effect of the source of nitrogen nutrition (nitrogen fixation or nitrate assimilation) on the response of pea plants to a gradual and moderate water stress was studied. Growth declined under water deficit, but nodulated plants were less sensitive to drought than nitrate-fed plants. Stomatal conductance and internal CO2 concentration also decreased, but both were higher in nitrogen-fixing plants throughout the drought period, leading to better maintenance of carbon assimilation rates under water deficit. Glycolate oxidase, a key enzyme in the photorespiratory cycle, declined by 50% in nitrogen-fixing plants under water deficit, although it was not affected in nitrate-fed plants. Nitrogen assimilation declined during the drought period and was independent of nitrogen source. Free amino acid content declined in leaves of plants grown under both nutrition regimes, reflecting the decrease in nitrogen assimilation. Water stress led to carbohydrate accumulation in pea plants grown with either nitrogen source, but it was higher in nitrogen-fixing plants. Roots showed the greatest carbohydrate and amino acid accumulation in both nutritions regimes, with significantly greater increases in free amino acids in nitrate-fed plants. It is concluded that the nitrogen source is a major factor affecting pea responses to water stress, although the difference in sensitivity seems to be related not to the nitrogen assimilation process but to complex interactions with photorespiratory flux and stomatal conductance.

The effect of the source of nitrogen (N) nutrition [N2 fixation and nitrate (NO3) assimilation] on plant productivity, gas exchange, N assimilation, water relations, and glycollate oxidase activity were studied in pea plants. Nitrogen‐fixing plants showed a higher RGR over the study period. This higher RGR was correlated with higher stomatal conductance and, consequently, a higher transpiration rate. Photosynthetic N‐use efficiency was also higher when plants were nodulated. We found glycollate oxidase activity, a key enzyme of the photorespiratory pathway, much lower in N2 fixing plants, what strongly suggests that energy losses due to photorespiration are lower in these plants, and therefore, photosynthetic efficiency was higher.

Summary Imazethapyr (IM) is a herbicide that inhibits the branched-chain amino acid (BCAA) biosynthesis through the specific inhibition of acetolactate synthase activity. This herbicide acts very slowly and several weeks are required for complete plant death. From the BCAA biosynthesis inhibition to the growth inhibition and plant death, the processes involved are not fully understood. Starvation for BCAAs and/or starvation for carbohydrates in sinks. have been proposed as part of the death mechanisms. In this study, a permanent acetolactate synthase inhibition is used in order to (1) determine whether the growth inhibition effects can be attributed to a reduction in BCAA content and/or to starvation of carbohydrates; and (2) to analyse the physiological changes induced. Sublethal doses of IM were continuously supplied in the nutrient solution of nodulated pea plants. These conditions led to a significant decline in plant growth. The herbicide also caused a decline in nodule initiation, but had little effect on nodule development. However, plants were not nitrogen-limited and net photosynthesis was only slightly affected at the higher herbicide concentration. Total soluble sugars and starch were accumulated in both leaves and roots following herbicide supply. These results were also found in non-nodulated, nitrate-fed plants. In relation with a likely BCAA starvation, a significant increase was observed in the free amino acid pool, with a marked imbalance among different amino acids, although among BCAAs, only valine pool declined as a consequence of IM supply. It is concluded that acetolactate synthase inhibition by continuous, sublethal IM supply does not induce carbohydrate or a specific BCAA starvation in pea plants.

Several ethyl 2,3-dihydro-3-oxoisothiazolo[5,4-b]pyridine-2-alkanoate derivatives were synthesized as herbicides. Only 5-methyl derivatives inhibited both hypocotyl and root growth in the lettuce (Lactuca sativa L.) seedling test at 100 mg litre -1 . Only ethyl propionate and valerate derivatives showed significant inhibition at 0.1 mg litre -1 , whereas ethyl acetate or butyrate derivatives were inactive. Contrary to unoxidized derivatives, the inhibitory effect of 1-oxide and 1,1-dioxide derivatives was strongly dependent on concentration; ethyl 2,3-dihydro-5-methyl-3-oxoisothiazolo[5,4-b]pyridine-2-propionate 1,1-dioxide inhibited 100% of germination at 100 mg litre -1 and 45% of lettuce seedling growth at 0.1 mg litre -1 . Quantitative structure-inhibition of growth relationship analysis carried out by adaptive least-squares (ALS) method gave a good correlation with small and hydrophobic 5-substituents as well as with odd carbon-chain ethyl alkanoates in position 2. Active compounds did not show auxin-like activity from 0.1 to 100 mg litre -1 .