Ana Zabalza

Nitrogen Fixation Control under Drought Stress. Localized or Systemic

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodules enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially droughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal.

Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes.

Evolutionary diversity can be driven by the interaction of plants with different environments. Molecular bases involved in ecological adaptations to abiotic constraints can be explored using genomic tools. Legumes are major crops worldwide and soil salinity is a main stress affecting yield in these plants. We analyzed in the Medicago truncatula legume the root transcriptome of two genotypes having contrasting responses to salt stress: TN1.11, sampled in a salty Tunisian soil, and the reference Jemalong A17 genotype. TN1.11 plants show increased root growth under salt stress as well as a differential accumulation of sodium ions when compared to A17. Transcriptomic analysis revealed specific gene clusters preferentially regulated by salt in root apices of TN1.11, notably those related to the auxin pathway and to changes in histone variant isoforms. Many genes encoding transcription factors (TFs) were also differentially regulated between the two genotypes in response to salt. Among those selected for functional studies, overexpression in roots of the A17 genotype of the bHLH-type TF most differentially regulated between genotypes improved significantly root growth under salt stress. Despite the global complexity of the differential transcriptional responses, we propose that an increase in this bHLH TF expression may be linked to the adaptation of M. truncatula to saline soil environments.

Changes in mitochondrial electron partitioning in response to herbicides inhibiting branched-chain amino acid biosynthesis in soybean.

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.

The possible role of quinate in the mode of action of glyphosate and acetolactate synthase inhibitors

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.

Impairment of carbon metabolism induced by the herbicide glyphosate

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.

Protein kinase GCN2 mediates responses to glyphosate in Arabidopsis.

BackgroundThe 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.ResultsIn 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.ConclusionsThese 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.

Role of exogenously supplied ferulic and p-coumaric acids in mimicking the mode of action of acetolactate synthase inhibiting herbicides.

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.

Branched-chain amino acid biosynthesis inhibitors: herbicide efficacy is associated with an induced carbon-nitrogen imbalance.

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.

The pattern of shikimate pathway and phenylpropanoids after inhibition by glyphosate or quinate feeding in pea roots

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.

Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots

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.