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1.
New Phytol ; 238(4): 1431-1445, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36840421

RESUMEN

Hydrogen sulfide is a signaling molecule in plants that regulates essential biological processes through protein persulfidation. However, little is known about sulfide-mediated regulation in relation to photorespiration. Here, we performed label-free quantitative proteomic analysis and observed a high impact on protein persulfidation levels when plants grown under nonphotorespiratory conditions were transferred to air, with 98.7% of the identified proteins being more persulfidated under suppressed photorespiration. Interestingly, a higher level of reactive oxygen species (ROS) was detected under nonphotorespiratory conditions. Analysis of the effect of sulfide on aspects associated with non- or photorespiratory growth conditions has demonstrated that it protects plants grown under suppressed photorespiration. Thus, sulfide amends the imbalance of carbon/nitrogen and restores ATP levels to concentrations like those of air-grown plants; balances the high level of ROS in plants under nonphotorespiratory conditions to reach a cellular redox state similar to that in air-grown plants; and regulates stomatal closure, to decrease the high guard cell ROS levels and induce stomatal aperture. In this way, sulfide signals the CO2 -dependent stomata movement, in the opposite direction of the established abscisic acid-dependent movement. Our findings suggest that the high persulfidation level under suppressed photorespiration reveals an essential role of sulfide signaling under these conditions.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Sulfuro de Hidrógeno , Arabidopsis/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteómica , Proteínas de Arabidopsis/metabolismo , Sulfuro de Hidrógeno/metabolismo , Sulfuros/farmacología , Sulfuros/metabolismo , Estrés Oxidativo , Plantas/metabolismo , Estomas de Plantas/fisiología
2.
J Exp Bot ; 74(19): 6023-6039, 2023 10 13.
Artículo en Inglés | MEDLINE | ID: mdl-37486799

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/metabolismo
3.
Physiol Plant ; 175(6): e14084, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38148200

RESUMEN

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/genética
4.
Physiol Plant ; 166(1): 240-250, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30628087

RESUMEN

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 , Temperatura
5.
BMC Genomics ; 18(1): 781, 2017 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-29025409

RESUMEN

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 radiación
6.
Plant Cell Physiol ; 58(12): 2112-2125, 2017 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-29059445

RESUMEN

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 Fisiológico
7.
J Exp Bot ; 67(10): 3095-108, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27117340

RESUMEN

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 Polimerasa
8.
J Integr Plant Biol ; 58(1): 48-66, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25869057

RESUMEN

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 & desarrollo
9.
Plant Physiol ; 162(4): 1834-48, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23743713

RESUMEN

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 , Transcriptoma
10.
J Exp Bot ; 65(19): 5557-66, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24948681

RESUMEN

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 , Transcriptoma
11.
Plant Cell Physiol ; 54(1): 107-18, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23161854

RESUMEN

The physiological role of K(+)-dependent and K(+)-independent asparaginases in plants remains unclear, and the contribution from individual isoforms during development is poorly understood. We have used reverse genetics to assess the phenotypes produced by the deficiency of K(+)-dependent NSE1 asparaginase in the model legume Lotus japonicus. For this purpose, four different mutants were identified by TILLING and characterized, two of which affected the structure and function of the asparaginase molecule and caused asparagine accumulation. Plant growth and total seed weight of mature mutant seeds as well as the level of both legumin and convicilin seed storage proteins were affected in the mutants. The mutants isolated in the present work are the first of their type in legumes and have enabled us to demonstrate the importance of asparagine and K(+)-dependent NSE1 asparaginase for nitrogen remobilization and seed production in L. japonicus plants.


Asunto(s)
Asparaginasa/metabolismo , Lotus/enzimología , Lotus/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Asparaginasa/química , Asparaginasa/genética , Asparagina/metabolismo , Mutación , Nitrógeno/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Almacenamiento de Semillas/genética , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo
12.
Mol Plant Microbe Interact ; 25(2): 211-9, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22007601

RESUMEN

Two photorespiratory mutants of Lotus japonicus deficient in plastid glutamine synthetase (GS(2)) were examined for their capacity to establish symbiotic association with Mesorhizobium loti bacteria. Biosynthetic glutamine synthetase (GS) activity was reduced by around 40% in crude nodule extracts from mutant plants as compared with the wild type (WT). Western blot analysis further confirmed the lack of GS(2) polypeptide in mutant nodules. The decrease in GS activity affected the nodular carbon metabolism under high CO(2) (suppressed photorespiration) conditions, although mutant plants were able to form nodules and fix atmospheric nitrogen. However, when WT and mutant plants were transferred to an ordinary air atmosphere (photorespiratory active conditions) the nodulation process and nitrogen fixation were substantially affected, particularly in mutant plants. The number and fresh weight of mutant nodules as well as acetylene reduction activity showed a strong inhibition compared with WT plants. Optical microscopy studies from mutant plant nodules revealed the anticipated senescence phenotype linked to an important reduction in starch and sucrose levels. These results show that, in Lotus japonicus, photorespiration and, particularly, GS(2) deficiency result in profound limitations in carbon metabolism that affect the nodulation process and nitrogen fixation.


Asunto(s)
Glutamato-Amoníaco Ligasa/metabolismo , Lotus/enzimología , Mesorhizobium/fisiología , Plastidios/enzimología , Nódulos de las Raíces de las Plantas/enzimología , Carbohidratos/análisis , Carbono/metabolismo , Respiración de la Célula , Regulación de la Expresión Génica de las Plantas/fisiología , Glutamato-Amoníaco Ligasa/genética , Isoenzimas , Lotus/genética , Lotus/microbiología , Lotus/ultraestructura , Mutación , Nitrógeno/metabolismo , Fijación del Nitrógeno/fisiología , Fenotipo , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/microbiología , Nódulos de las Raíces de las Plantas/ultraestructura , Simbiosis
13.
Int J Mol Sci ; 13(7): 7994-8024, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22942686

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 Proteica
14.
Planta ; 234(1): 109-22, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21390508

RESUMEN

The molecular features responsible for the existence in plants of K+-dependent asparaginases have been investigated. For this purpose, two different cDNAs were isolated in Lotus japonicus, encoding for K+-dependent (LjNSE1) or K+-independent (LjNSE2) asparaginases. Recombinant proteins encoded by these cDNAs have been purified and characterized. Both types of asparaginases are composed by two different subunits, α (20 kDa) and ß (17 kDa), disposed as (αß)2 quaternary structure. Major differences were found in the catalytic efficiency of both enzymes, due to the fact that K+ is able to increase by tenfold the enzyme activity and lowers the K(m) for asparagine specifically in LjNSE1 but not in LjNSE2 isoform. Optimum LjNSE1 activity was found at 5-50 mM K+, with a K(m) for K+ of 0.25 mM. Na+ and Rb+ can, to some extent, substitute for K+ on the activating effect of LjNSE1 more efficiently than Cs+ and Li+ does. In addition, K+ is able to stabilize LjNSE1 against thermal inactivation. Protein homology modelling and molecular dynamics studies, complemented with site-directed mutagenesis, revealed the key importance of E248, D285 and E286 residues for the catalytic activity and K+ dependence of LjNSE1, as well as the crucial relevance of K+ for the proper orientation of asparagine substrate within the enzyme molecule. On the other hand, LjNSE2 but not LjNSE1 showed ß-aspartyl-hydrolase activity (K(m) = 0.54 mM for ß-Asp-His). These results are discussed in terms of the different physiological significance of these isoenzymes in plants.


Asunto(s)
Asparaginasa/metabolismo , Lotus/enzimología , Lotus/genética , Potasio/metabolismo , Secuencia de Aminoácidos , Aminoácidos/fisiología , Asparaginasa/química , Asparaginasa/genética , Secuencia de Bases , ADN Complementario/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Isoenzimas , Lotus/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nitrógeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad
15.
Planta ; 231(5): 1101-11, 2010 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-20237895

RESUMEN

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 Tiempo
16.
New Phytol ; 188(4): 1001-13, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20796214

RESUMEN

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 Genética
17.
Plants (Basel) ; 9(6)2020 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-32575698

RESUMEN

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.

18.
J Plant Physiol ; 236: 88-95, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30939333

RESUMEN

Enhanced ultraviolet radiation (UV) is an important environmental factor that may cause reductions in the growth and productivity of plants. In the present work we studied the response to UV-B radiation in leaves of the model legume Lotus japonicus. After UV-B treatment, induction of phenyalanine-ammonia lyase gene expression and enzyme activity was detected. Among the ten genes encoding for PAL found in the L. japonicus genome, LjPAL1 was both the most expressed and the most induced. All the genes encoding for enzymes of the isoflavonoid pathway were also strongly induced; this was paralleled by a marked accumulation of vestitol and isoliquiritigenin. Moreover, accumulation of several other isoflavonoids was also detected. In vitro measurements of the free radical scavenging capacity of vestitol indicated that this compound can be an appropriate free radical scavenger, suggesting a possible role for this molecule in the response to abiotic stress. On the other hand, an increase of flavonol levels was not observed while the expression of the key enzymes for flavonol biosynthesis flavanone-3-hydroxylase and flavonol synthase was decreased. Taken together, these results indicate that L. japonicus follows a peculiar strategy in its response to UV radiation by accumulating isoflavonoids as an possible alternative to accumulation of flavonols as observed in other plant species.


Asunto(s)
Isoflavonas/biosíntesis , Lotus/efectos de la radiación , Cromatografía Líquida de Alta Presión , Inducción Enzimática/efectos de la radiación , Depuradores de Radicales Libres/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Lotus/metabolismo , Espectrometría de Masas , Fenilanina Amoníaco-Liasa/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Reacción en Cadena en Tiempo Real de la Polimerasa , Rayos Ultravioleta
19.
Mol Plant Microbe Interact ; 19(1): 80-91, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16404956

RESUMEN

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.


Asunto(s)
Mapeo Cromosómico , Genes de Plantas/genética , Lotus/genética , Simbiosis/genética , Alelos , Ligamiento Genético , Marcadores Genéticos , Genoma de Planta , Repeticiones de Microsatélite , Mutación/genética , Fenotipo , Recombinación Genética
20.
PLoS One ; 11(5): e0156568, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27233042

RESUMEN

[This corrects the article DOI: 10.1371/journal.pone.0130438.].

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