Carmen M. Pérez-Delgado

Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism.

Cellular Stress Following Water Deprivation in the Model Legume Lotus japonicus

Drought stress is one of the most important factors in the limitation of plant productivity worldwide. In order to cope with water deprivation, plants have adopted several strategies that produce major changes in gene expression. In this paper, the response to drought stress in the model legume Lotus japonicus was studied using a transcriptomic approach. Drought induced an extensive reprogramming of the transcriptome as related to various aspects of cellular metabolism, including genes involved in photosynthesis, amino acid metabolism and cell wall metabolism, among others. A particular focus was made on the genes involved in the cellular stress response. Key genes involved in the control of the cell cycle, antioxidant defense and stress signaling, were modulated as a consequence of water deprivation. Genes belonging to different families of transcription factors were also highly responsive to stress. Several of them were homologies to known stress-responsive genes from the model plant Arabidopsis thaliana, while some novel transcription factors were peculiar to the L. japonicus drought stress response.

Transcriptomic and Metabolic Changes Associated with Photorespiratory Ammonium Accumulation in the Model Legume Lotus japonicus

Transcriptomic and metabolomic analysis of a plastidic glutamine synthetase mutant shows a coordinate repression of photorespiratory genes and accumulation of several key metabolites. The transcriptomic and metabolic consequences of the lack of plastidic glutamine (Gln) synthetase in the model legume Lotus japonicus were investigated. Wild-type and mutant plants lacking the plastidic isoform of Gln synthetase were grown in conditions that suppress photorespiration and then transferred for different lengths of time to photorespiratory conditions. Transcript and metabolite levels were determined at the different time points considered. Under photorespiratory active conditions, the mutant accumulated high levels of ammonium, followed by its subsequent decline. A coordinate repression of the photorespiratory genes was observed in the mutant background. This was part of a greater modulation of the transcriptome, especially in the mutant, that was paralleled by changes in the levels of several key metabolites. The data obtained for the mutant represent the first direct experimental evidence for a coordinate regulation of photorespiratory genes over time. Metabolomic analysis demonstrated that mutant plants under active photorespiratory conditions accumulated high levels of several amino acids and organic acids, including intermediates of the Krebs cycle. An increase in Gln levels was also detected in the mutant, which was paralleled by an increase in cytosolic Gln synthetase1 gene transcription and enzyme activity levels. The global panoramic of the transcripts and metabolites that changed in L. japonicus plants during the transfer from photorespiration-suppressed to photorespiration-active conditions highlighted the link between photorespiration and several other cellular processes, including central carbon metabolism, amino acid metabolism, and secondary metabolism.

Reassimilation of ammonium in Lotus japonicus

This review summarizes the most recent results obtained in the analysis of two important metabolic pathways involved in the release of internal sources of ammonium in the model legume Lotus japonicus: photorespiratory metabolism and asparagine breakdown mediated by aparaginase (NSE). The use of photorespiratory mutants deficient in plastidic glutamine synthetase (GS2) enabled us to investigate the transcriptomics and metabolomic changes associated with photorespiratory ammonium accumulation in this plant. The results obtained indicate the existence of a coordinate regulation of genes involved in photorespiratory metabolism. Other types of evidence illustrate the multiple interconnections existing among the photorespiratory pathway and other processes such as intermediate metabolism, nodule function, and secondary metabolism in this plant, all of which are substantially affected in GS2-deficient mutants because of the impairment of the photorespiratory cycle. Finally, the importance of asparagine metabolism in L. japonicus is highlighted because of the fact that asparagine constitutes the vast majority of the reduced nitrogen translocated between different organs of this plant. The different types of NSE enzymes and genes which are present in L. japonicus are described. There is a particular focus on the most abundant K(+)-dependent LjNSE1 isoform and how TILLING mutants were used to demonstrate by reverse genetics the importance of this particular isoform in plant growth and seed production.

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

It is well established that the plastidic isoform of glutamine synthetase (GS2) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH4 + accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS2. The mutant plants accumulated high levels of NH4 + when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS2 that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH4 +. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS1 in the mutant, thus revealing a major role of cytosolic GS1 in the reassimilation and detoxification of photorespiratory NH4 + when the plastidic GS2 isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.

Modulation of phenolic metabolism under stress conditions in a Lotus japonicus mutant lacking plastidic glutamine synthetase

This paper was aimed to investigate the possible implications of the lack of plastidic glutamine synthetase (GS2) in phenolic metabolism during stress responses in the model legume Lotus japonicus. Important changes in the transcriptome were detected in a GS2 mutant called Ljgln2-2, compared to the wild type, in response to two separate stress conditions, such as drought or the result of the impairment of the photorespiratory cycle. Detailed transcriptomic analysis showed that the biosynthesis of phenolic compounds was affected in the mutant plants in these two different types of stress situations. For this reason, the genes and metabolites related to this metabolic route were further investigated using a combined approach of gene expression analysis and metabolite profiling. A high induction of the expression of several genes for the biosynthesis of different branches of the phenolic biosynthetic pathway was detected by qRT-PCR. The extent of induction was always higher in Ljgln2-2, probably reflecting the higher stress levels present in this genotype. This was paralleled by accumulation of several kaempferol and quercetine glycosides, some of them described for the first time in L. japonicus, and of high levels of the isoflavonoid vestitol. The results obtained indicate that the absence of GS2 affects different aspects of phenolic metabolism in L. japonicus plants in response to stress.

Amino Acids and Drought Stress in Lotus: Use of Transcriptomics and Plastidic Glutamine Synthetase Mutants for New Insights in Proline Metabolism

SECTION I C INTRODUCTION 1. Environmental Change, and Plant Amino Acids and Their Derivatives - An Introduction SECTION II C AMINO ACIDS AND PEPTIDES, AND PLANT STRESS ADAPTATION 2. 5-Aminolevulinic Acid GBP5-ALA- A Multifunctional Amino Acid as A Plant Growth Stimulator and Stress Tolerance Factor 3. Cysteine - Jack of All Glutathione-based Plant Stress Defense Trades 4. Amino Acids and Drought Stress in Lotus: Use of Transcriptomics and Plastidic Glutamine Synthetase Mutants for New Insights in Proline Metabolism 5. Modulation of Proline - Implications in Plant Stress Tolerance and Development 6. Target Osmoprotectants for Abiotic Stress Tolerance in Crop Plants - Glycine Betaine and Proline SECTION III C AMINES AND BRASSINOSTEROIDS, AND PLANT STRESS ADAPTATION 7. Polyamines as Indicators and as Modulators in the Abiotic Stress in Plants 8. Polyamines in Stress Protection: Applications in Agriculture 9. Functional Role of Polyamines and Polyamine-Metabolizing Enzymes during Salinity, Drought and Cold stresses 10. Regulatory Role of Polyamines in Growth, Development and Abiotic Stress Tolerance in Plants 11. Polyamines - Involvement in Plant Stress Tolerance and Adaptation 12. Role of Polyamines in Plant-Pathogen Interactions 13. Role of Polyamines in Stress Management 14. Polyamines in Plant In Vitro Culture 15. Betaines and Related Osmoprotectants - Significance in Metabolic Engineering of Plant Stress Resistance 16. Brassinosteroids Role for Amino Acids, Peptides and Amines Modulation in Stressed Plants C A Review SECTION IV C APPRAISAL AND PERSPECTIVES 17. Plant Adaptation to Environmental Change and Significance of Amino Acids and Their Derivatives C Appraisal and Perspectives

Use of transcriptomics and co-expression networks to analyze the interconnections between nitrogen assimilation and photorespiratory metabolism

Highlight A clear interconnection between photorespiration and primary nitrogen assimilation is established in Lotus japonicus, and key transcription factors connected to both routes are identified using transcriptomics and gene co-expression networks.

Elevated CO2 Induces Root Defensive Mechanisms in Tomato Plants When Dealing with Ammonium Toxicity

An adequate carbon supply is fundamental for plants to thrive under ammonium stress. In this work, we studied the mechanisms involved in tomato (Solanum lycopersicum L.) response to ammonium toxicity when grown under ambient or elevated CO2 conditions (400 or 800 p.p.m. CO2). Tomato roots were observed to be the primary organ dealing with ammonium nutrition. We therefore analyzed nitrogen (N) and carbon (C) metabolism in the roots, integrating the physiological response with transcriptomic regulation. Elevated levels of CO2 preferentially stimulated root growth despite the high ammonium content. The induction of anaplerotic enzymes from the tricarboxylic acid (TCA) cycle led to enhanced amino acid synthesis under ammonium nutrition. Furthermore, the root transcriptional response to ammonium toxicity was improved by CO2-enriched conditions, leading to higher expression of stress-related genes, as well as enhanced modulation of genes related to signaling, transcription, transport and hormone metabolism. Tomato roots exposed to ammonium stress also showed a defense-like transcriptional response according to the modulation of genes related to detoxification and secondary metabolism, involving principally terpenoid and phenolic compounds. These results indicate that increasing C supply allowed the co-ordinated regulation of root defense mechanisms when dealing with ammonium toxicity.

Expression, activity of phenylalanine-ammonia-lyase and accumulation of phenolic compounds in Lotus japonicus under salt stress

Abstract In the present work the gene expression levels and enzyme activity of phenylalanine-ammonia-lyase (PAL) were investigated in plants of the model legume Lotus japonicus under control and stress conditions. PAL is the key enzyme of the phenylpropanoid pathway, a route that involves numerous branches that lead to the production of a wide range of metabolites that can play an important role in the response to different kind of environmental stress. Unexpectedly treatment with salt caused decrease in activity of PAL in roots and the expression of several isogenes for PAL was significantly altered in roots or leaves; although the most highly expressed one, LjPAL10, remained unaffected by the treatment. Changes in tissue contents of some phenolic compounds, namely increase of quercetin glycosides and decrease of some phenylpropanoids were observed. The possible physiological meaning of the accumulation of phenolic compounds together with the changes in PAL enzyme activity is discussed.