RESUMEN
Photorespiration has been considered a 'futile' cycle in C3 plants, necessary to detoxify and recycle the metabolites generated by the oxygenating activity of Rubisco. However, several reports indicate that this metabolic route plays a fundamental role in plant metabolism and constitutes a very interesting research topic. Many open questions still remain with regard to photorespiration. One of these questions is how the photorespiratory process is regulated in plants and what factors contribute to this regulation. In this review, we summarize recent advances in the regulation of the photorespiratory pathway with a special focus on the transcriptional and post-translational regulation of photorespiration and the interconnections of this process with nitrogen and sulfur metabolism. Recent findings on sulfide signaling and protein persulfidation are also described.
Asunto(s)
Fotosíntesis , Plantas , Fotosíntesis/fisiología , Plantas/genética , Plantas/metabolismo , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
Isoflavonoids are mostly produced by legumes although little is known about why and how legumes are able to regulate the biosynthesis of these particular compounds. Understanding the role of potential regulatory genes of the isoflavonoid biosynthetic pathway constitutes an important topic of research. The LORE1 mutation of the gene encoding the transcription factor MYB36 allowed the identification of this gene as a regulator of isoflavonoid biosynthesis in Lotus japonicus plants. The levels of several isoflavonoid compounds were considerably lower in two lines of Ljmyb36 mutant plants compared to the WT. In addition, we found that Ljmyb36 mutant plants were significantly smaller and showed a substantial decrease in the chlorophyll levels under normal growth conditions. The analysis of plants subjected to different types of abiotic stress conditions further revealed that mutant plants presented a higher sensitivity than WT plants, indicating that the MYB36 transcription factor is also involved in the stress response in L. japonicus plants.
Asunto(s)
Lotus , Lotus/genética , Lotus/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Mutación/genética , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
Agriculture faces the considerable challenge of having to adapt to a progressively changing climate (including the increase in CO2 levels and temperatures); environmental impact must be reduced while at the same time crop yields need to be maintained or increased to ensure food security. Under this scenario, increasing plants' nitrogen (N) use efficiency and minimizing the energy losses associated with photorespiration are two goals of crop breeding that are long sought after. The plastidic glutamine synthetase (GS2) enzyme stands at the crossroads of N assimilation and photorespiration, and is therefore a key candidate for the improvement of crop performance. The GS2 enzyme has long been considered essential for angiosperm survival under photorespiratory conditions. Surprisingly, in Arabidopsis GS2 is not essential for plant survival, and its absence confers tolerance towards ammonium stress, which is in conflict with the idea that NH4+ accumulation is one of the main causes of ammonium stress. Altogether, it appears that the 'textbook' view of this enzyme must be revisited, especially regarding the degree to which it is essential for plant growth under photorespiratory conditions, and the role of NH4+ assimilation during ammonium stress. In this article we open the debate on whether more or less GS2 is a desirable trait for plant productivity.
Asunto(s)
Compuestos de Amonio , Arabidopsis , Tracheophyta , Arabidopsis/genética , Cycadopsida , Glutamato-Amoníaco Ligasa/genética , Nitrógeno , Fitomejoramiento , PlastidiosRESUMEN
The afterglow (AG) luminescence is a delayed chlorophyll fluorescence emitted by the photosystem II that seems to reflect the level of assimilatory potential (NADPH+ATP) in chloroplast. In this work, the thermoluminescence (TL) emissions corresponding to the AG band were investigated in plants of the WT and the Ljgln2-2 photorespiratory mutant from Lotus japonicus grown under either photorespiratory (air) or non-photorespiratory (high concentration of CO2 ) conditions. TL glow curves obtained after two flashes induced the strongest overall TL emissions, which could be decomposed in two components: B band (tmax = 27-29°C) and AG band (tmax = 44-45°C). Under photorespiratory conditions, WT plants showed a ratio of 1.17 between the intensity of the AG and B bands (IAG /IB ). This ratio increased considerably under non-photorespiratory conditions (2.12). In contrast, mutant Ljgln2-2 plants grown under both conditions showed a high IAG /IB ratio, similar to that of WT plants grown under non-photorespiratory conditions. In addition, high temperature thermoluminescence (HTL) emissions associated to lipid peroxidation were also recorded. WT and Ljgln2-2 mutant plants grown under photorespiratory conditions showed both a significant HTL band, which increased significantly under non-photorespiratory conditions. The results of this work indicate that changes in the amplitude of IAG /IB ratio could be used as an in vivo indicator of alteration in the level of photorespiratory metabolism in L. japonicus chloroplasts. Moreover, the HTL results suggest that photorespiration plays some role in the protection of the chloroplast against lipid peroxidation.
Asunto(s)
Lotus/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Cloroplastos/metabolismo , Transporte de Electrón/fisiología , Luminiscencia , TemperaturaRESUMEN
BACKGROUND: Asparagine is a very important nitrogen transport and storage compound in plants due to its high nitrogen/carbon ratio and stability. Asparagine intracellular concentration depends on a balance between asparagine biosynthesis and degradation. The main enzymes involved in asparagine metabolism are asparagine synthetase (ASN), asparaginase (NSE) and serine-glyoxylate aminotransferase (SGAT). The study of the genes encoding for these enzymes in the model legume Lotus japonicus is of particular interest since it has been proposed that asparagine is the principal molecule used to transport reduced nitrogen within the plant in most temperate legumes. RESULTS: A differential expression of genes encoding for several enzymes involved in asparagine metabolism was detected in L. japonicus. ASN is encoded by three genes, LjASN1 was the most highly expressed in mature leaves while LjASN2 expression was negligible and LjASN3 showed a low expression in this organ, suggesting that LjASN1 is the main gene responsible for asparagine synthesis in mature leaves. In young leaves, LjASN3 was the only ASN gene expressed although at low levels, while all the three genes encoding for NSE were highly expressed, especially LjNSE1. In nodules, LjASN2 and LjNSE2 were the most highly expressed genes, suggesting an important role for these genes in this organ. Several lines of evidence support the connection between asparagine metabolic genes and photorespiration in L. japonicus: a) a mutant plant deficient in LjNSE1 showed a dramatic decrease in the expression of the two genes encoding for SGAT; b) expression of the genes involved in asparagine metabolism is altered in a photorespiratory mutant lacking plastidic glutamine synthetase; c) a clustering analysis indicated a similar pattern of expression among several genes involved in photorespiratory and asparagine metabolism, indicating a clear link between LjASN1 and LjSGAT genes and photorespiration. CONCLUSIONS: The results obtained in this paper indicate the existence of a differential expression of asparagine metabolic genes in L. japonicus and point out the crucial relevance of particular genes in different organs. Moreover, the data presented establish clear links between asparagine and photorespiratory metabolic genes in this plant.
Asunto(s)
Asparagina/metabolismo , Respiración de la Célula/efectos de la radiación , Perfilación de la Expresión Génica , Luz , Lotus/genética , Lotus/metabolismo , Respiración de la Célula/genética , Lotus/efectos de la radiación , Mutación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de la radiaciónRESUMEN
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.
Asunto(s)
Compuestos de Amonio/toxicidad , Dióxido de Carbono/metabolismo , Raíces de Plantas/metabolismo , Solanum lycopersicum/fisiología , Compuestos de Amonio/metabolismo , Biomasa , Carbono/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Solanum lycopersicum/efectos de los fármacos , Nitratos/metabolismo , Nitratos/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Estrés FisiológicoRESUMEN
New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15-24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text].
Asunto(s)
Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Pirimidinas/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A1/química , Antagonistas del Receptor de Adenosina A2/síntesis química , Antagonistas del Receptor de Adenosina A2/química , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Photorespiration is an essential high flux metabolic pathway that is found in all oxygen-producing photosynthetic organisms. It is often viewed as a closed metabolic repair pathway that serves to detoxify 2-phosphoglycolic acid and to recycle carbon to fuel the Calvin-Benson cycle. However, this view is too simplistic since the photorespiratory cycle is known to interact with several primary metabolic pathways, including photosynthesis, nitrate assimilation, amino acid metabolism, C1 metabolism and the Krebs (TCA) cycle. Here we will review recent advances in photorespiration research and discuss future priorities to better understand (i) the metabolic integration of the photorespiratory cycle within the complex network of plant primary metabolism and (ii) the importance of photorespiration in response to abiotic and biotic stresses.
Asunto(s)
Fotosíntesis/fisiología , Plantas/metabolismo , Redes y Vías Metabólicas/fisiología , Fenómenos Fisiológicos de las Plantas , Plantas/enzimologíaRESUMEN
Nitrogen is one of the most important nutrients for plants and, in natural soils, its availability is often a major limiting factor for plant growth. Here we examine the effect of different forms of nitrogen nutrition and of photorespiration on gene expression in the model legume Lotus japonicus with the aim of identifying regulatory candidate genes co-ordinating primary nitrogen assimilation and photorespiration. The transcriptomic changes produced by the use of different nitrogen sources in leaves of L. japonicus plants combined with the transcriptomic changes produced in the same tissue by different photorespiratory conditions were examined. The results obtained provide novel information on the possible role of plastidic glutamine synthetase in the response to different nitrogen sources and in the C/N balance of L. japonicus plants. The use of gene co-expression networks establishes a clear relationship between photorespiration and primary nitrogen assimilation and identifies possible transcription factors connected to the genes of both routes.
Asunto(s)
Perfilación de la Expresión Génica , Nitrógeno/metabolismo , Fotosíntesis/fisiología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas/fisiología , Genes de Plantas/genética , Genes de Plantas/fisiología , Glutamato-Amoníaco Ligasa/metabolismo , Lotus/genética , Lotus/metabolismo , Lotus/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Recycling of the 2-phosphoglycolate generated by the oxygenase reaction of Rubisco requires a complex and energy-consuming set of reactions collectively known as the photorespiratory cycle. Several approaches aimed at reducing the rates of photorespiratory energy or carbon loss have been proposed, based either on screening for natural variation or by means of genetic engineering. Recent work indicates that plant yield can be substantially improved by the alteration of photorespiratory fluxes or by engineering artificial bypasses to photorespiration. However, there is also evidence indicating that, under certain environmental and/or nutritional conditions, reduced photorespiratory capacity may be detrimental to plant performance. Here we summarize recent advances obtained in photorespiratory engineering and discuss prospects for these advances to be transferred to major crops to help address the globally increasing demand for food and biomass production.
Asunto(s)
Producción de Cultivos/métodos , Ingeniería Genética/métodos , Fotosíntesis/genética , Dióxido de Carbono/metabolismo , Fotosíntesis/fisiología , Desarrollo de la Planta/genética , Plantas/genéticaRESUMEN
A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1-31) were synthesized to evaluate some structural modifications at the 2- and 5-positions aimed at shifting affinity towards the human (h) A2A adenosine receptor (AR) or both hA2A and hA1 ARs. The most active compounds were those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a substituted-benzyl group at position 2. Several of these derivatives (22-31) displayed nanomolar affinity for the hA2A AR (Ki=3.62-57nM) and slightly lower for the hA1 ARs, thus showing different degrees (3-22 fold) of hA2A versus hA1 selectivity. In particular, the 2-(2-methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 25 possessed the highest hA2A and hA1 AR affinities (Ki=3.62nM and 18nM, respectively) and behaved as potent antagonist at both these receptors (cAMP assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA2A AR (Ki=5.26nM) and was 22-fold selective versus the hA1 subtype. Molecular docking investigations performed at the hA2A AR crystal structure and at a homology model of the hA1 AR allowed us to represent the hypothetical binding mode of our derivatives and to rationalize the observed SARs.
Asunto(s)
Antagonistas de Receptores Purinérgicos P1/química , Antagonistas de Receptores Purinérgicos P1/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A2A/metabolismo , Aminación , Humanos , Simulación del Acoplamiento Molecular , Pirazoles/química , Pirazoles/farmacologíaRESUMEN
Elucidating the relationships between gene expression and the physiological mechanisms remains a bottleneck in breeding for resistance to salinity and drought. This study related the expression of key target genes with the physiological performance of durum wheat under different combinations of salinity and irrigation. The candidate genes assayed included two encoding for the DREB (dehydration responsive element binding) transcription factors TaDREB1A and TaDREB2B, another two for the cytosolic and plastidic glutamine synthetase (TaGS1 and TaGS2), and one for the specific Na(+) /H(+) vacuolar antiporter (TaNHX1). Expression of these genes was related to growth and different trait indicators of nitrogen metabolism (nitrogen content, stable nitrogen isotope composition, and glutamine synthetase and nitrate reductase activities), photosynthetic carbon metabolism (stable carbon isotope composition and different gas exchange traits) and ion accumulation. Significant interaction between genotype and growing conditions occurred for growth, nitrogen content, and the expression of most genes. In general terms, higher expression of TaGS1, TaGS2, TaDREB2B, and to a lesser extent of TaNHX1 were associated with a better genotypic performance in growth, nitrogen, and carbon photosynthetic metabolism under salinity and water stress. However, TaDREB1A was increased in expression under stress compared with control conditions, with tolerant genotypes exhibiting lower expression than susceptible ones.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Salinidad , Triticum/genética , Triticum/fisiología , Adaptación Fisiológica/genética , Biomasa , Carbono/metabolismo , Citosol/metabolismo , Deshidratación , Gases/metabolismo , Genes de Plantas , Genotipo , Iones , Nitrógeno/metabolismo , Péptidos/metabolismo , Fotosíntesis/genética , Transpiración de Plantas/genética , Carácter Cuantitativo Heredable , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estrés Fisiológico , Triticum/crecimiento & desarrolloRESUMEN
The 1,2,4-triazolo[1,5-a]quinoxaline (TQX) scaffold was extensively investigated in our previously reported studies and recently, our attention was focused at position 5 of the tricyclic nucleus where different acyl and carboxylate moieties were introduced (compounds 2-15). This study produced some interesting compounds endowed with good hA3 receptor affinity and selectivity. In addition, to find new insights about the structural requirements for hA3 receptor-ligand interaction, the tricyclic TQX ring was destroyed yielding some 1,2,4-triazole derivatives (compounds 16-23). These simplified compounds, though maintaining the crucial structural requirements for adenosine receptor-ligand interaction, have a very low hA3 adenosine receptor affinity, the only exception being compound 23 (1-[3-(4-methoxyphenyl)-1-phenyl-1H-1,2,4-triazol-5-yl]-3-phenylurea) endowed with a Ki value in the micro-molar range and high hA3 selectivity versus both hA1 and hA2A AR subtypes. Evaluation of the side products obtained in the herein reported synthetic pathways led to the identification of some new triazolo[1,5-a]quinoxalines as hA3AR antagonists (compounds 24-27). These derivatives, though lacking the classical structural requirements for the anchoring at the hA3 receptor site, show high hA3 affinity and in some case selectivity versus hA1 and hA2A subtypes. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA3 receptor.
Asunto(s)
Antagonistas del Receptor de Adenosina A3/química , Quinoxalinas/química , Antagonistas del Receptor de Adenosina A3/síntesis química , Animales , Sitios de Unión , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Ligandos , Modelos Moleculares , Quinoxalinas/síntesis química , Quinoxalinas/farmacología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Amoníaco/metabolismo , Glutamato-Amoníaco Ligasa/genética , Lotus/genética , Lotus/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Aminoácidos/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Ciclo del Ácido Cítrico/genética , Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Fotosíntesis/genética , Fotosíntesis/fisiología , Plastidios/genética , Plastidios/metabolismo , Metabolismo Secundario , TranscriptomaRESUMEN
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.
Asunto(s)
Compuestos de Amonio/metabolismo , Asparagina/metabolismo , Regulación de la Expresión Génica de las Plantas , Glutamato-Amoníaco Ligasa/metabolismo , Lotus/metabolismo , Nitrógeno/metabolismo , Transporte Biológico , Respiración de la Célula , Glutamato-Amoníaco Ligasa/genética , Luz , Lotus/genética , Lotus/efectos de la radiación , Estructura Molecular , Mutación , Plastidios/enzimología , Potasio/metabolismo , Genética Inversa , Semillas/genética , Semillas/metabolismo , Semillas/efectos de la radiación , TranscriptomaRESUMEN
In this work, we studied the combined effect of increased temperature and atmospheric CO2, salt and drought stress, and inoculation with plant-growth-promoting rhizobacteria (PGPR) on the growth and some nutritional parameters of the edible halophyte Salicornia ramosissima. We found that the increase in temperature and atmospheric CO2, combined with salt and drought stresses, led to important changes in S. ramosissima fatty acids (FA), phenols, and oxalate contents, which are compounds of great importance for human health. Our results suggest that the S. ramosissima lipid profile will change in a future climate change scenario, and that levels of oxalate and phenolic compounds may change in response to salt and drought stress. The effect of inoculation with PGPR depended on the strains used. Some strains induced the accumulation of phenols in S. ramosissima leaves at higher temperature and CO2 while not altering FA profile but also led to an accumulation of oxalate under salt stress. In a climate change scenario, a combination of stressors (temperature, salinity, drought) and environmental conditions (atmospheric CO2, PGPR) will lead to important changes in the nutritional profiles of edible plants. These results may open new perspectives for the nutritional and economical valorization of S. ramosissima.
RESUMEN
Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N(2) 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.
Asunto(s)
Fabaceae/enzimología , Glutamato-Amoníaco Ligasa/química , Proteínas de Plantas/química , Animales , Cristalografía por Rayos X , Genes de Plantas , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Humanos , Modelos Moleculares , Fijación del Nitrógeno , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/enzimología , Conformación ProteicaRESUMEN
We have studied the possible role, in a plant glutamine synthetase (GS), of the different cysteinyl residues present in this enzyme. For this purpose we carried out the site-directed mutagenesis of the cDNA for alpha-GS polypeptide from Phaseolus vulgaris in the positions corresponding to Cys-92, Cys-159, and Cys-179, followed by heterologous expression in E. coli and enzymatic characterisation of WT and mutant proteins. The results show that neither Cys-92 nor Cys-179 residues were essential for enzyme activity, but the replacement of Cys-159 by alanine or serine strongly affects the quaternary structure and function of the GS enzyme molecule, resulting in a complete loss of enzymatic activity. Other studies using sulfhydryl specific reagents such as pHMB (p-hydroxymercuribenzoate) or DTNB (5,5'-dithiobis-2-nitrobenzoate) confirmed that the profound inhibition produced is associated with an important alteration of the quaternary structure of GS, and suggest that Cys-159 might be the residue responsible for the enzyme inhibition. All these results suggest that the Cys-159 residue is essential for the enzyme structure. The results are also consistent with previous reports based on classical biochemistry studies indicating the presence of essential cysteinyl residues for the enzyme activity of higher plant GS.
Asunto(s)
Cisteína/metabolismo , Glutamato-Amoníaco Ligasa/química , Phaseolus/enzimología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Biocatálisis/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Escherichia coli , Glutamato-Amoníaco Ligasa/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Phaseolus/efectos de los fármacos , Estructura Secundaria de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/farmacología , Factores de TiempoRESUMEN
The role of plastidic glutamine synthetase (GS2) in proline biosynthesis and drought stress responses in Lotus japonicus was investigated using the GS2 mutant, Ljgln2-2. Wild-type (WT) and mutant plants were submitted to different lengths of time of water and nutrient solution deprivation. Several biochemical markers were measured and the transcriptional response to drought was determined by both quantitative real-time polymerase chain reaction and transcriptomics. The Ljgln2-2 mutant exhibited normal sensitivity to mild water deprivation, but physiological, biochemical and massive transcriptional differences were detected in the mutant, which compromised recovery (rehydration) following re-watering after severe drought stress. Proline accumulation during drought was substantially lower in mutant than in WT plants, and significant differences in the pattern of expression of the genes involved in proline metabolism were observed. Transcriptomic analysis revealed that about three times as many genes were regulated in response to drought in Ljgln2-2 plants compared with WT. The transcriptomic and accompanying biochemical data indicate that the Ljgln2-2 mutant is subject to more intense cellular stress than WT during drought. The results presented here implicate plastidic GS2 in proline production during stress and provide interesting insights into the function of proline in response to drought.
Asunto(s)
Sequías , Perfilación de la Expresión Génica , Glutamato-Amoníaco Ligasa/deficiencia , Lotus/genética , Lotus/metabolismo , Plastidios/enzimología , Prolina/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Cinética , Lotus/enzimología , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Plastidios/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estrés Fisiológico/genética , Transcripción GenéticaRESUMEN
Phenylpropanoid metabolism represents an important metabolic pathway from which originates a wide number of secondary metabolites derived from phenylalanine or tyrosine, such as flavonoids and isoflavonoids, crucial molecules in plants implicated in a large number of biological processes. Therefore, various types of interconnection exist between different aspects of nitrogen metabolism and the biosynthesis of these compounds. For legumes, flavonoids and isoflavonoids are postulated to play pivotal roles in adaptation to their biological environments, both as defensive compounds (phytoalexins) and as chemical signals in symbiotic nitrogen fixation with rhizobia. In this paper, we summarize the recent progress made in the characterization of flavonoid and isoflavonoid biosynthetic pathways in the model legume Lotus japonicus (Regel) Larsen under different abiotic stress situations, such as drought, the impairment of photorespiration and UV-B irradiation. Emphasis is placed on results obtained using photorespiratory mutants deficient in glutamine synthetase. The results provide different types of evidence showing that an enhancement of isoflavonoid compared to standard flavonol metabolism frequently occurs in Lotus under abiotic stress conditions. The advance produced in the analysis of isoflavonoid regulatory proteins by the use of co-expression networks, particularly MYB transcription factors, is also described. The results obtained in Lotus japonicus plants can be also extrapolated to other cultivated legume species, such as soybean, of extraordinary agronomic importance with a high impact in feeding, oil production and human health.