Genes for asparagine metabolism in Lotus japonicus : differential expression and interconnection with photorespiration

Background: Asparagine is a very important nitrogen transport and storage compound in plants due to its high nitrogen/carbon ratio and stability. Asparagine intracellu lar concentration depends on a balance between asparagine biosynthesis and degradation. The main enzymes involved in asparagine metabolism are as paragine synthetase (ASN), asparaginase (NSE) and serine-glyoxylate aminotransfera se (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 seve ral 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 .Innodules, 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 exis tence 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 betw een asparagine and photorespiratory metabolic genes in this plant.Junta de Andalucía (P10-CVI- 6368)FEDER-Ministerio de Economía y Competitividad (AGL 2014 – 54413-R

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 activityJunta de Andalucía P1OCVI- 6368 y BIO-163Ministerio de Economía y Competitividad AGL2014-54413-

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

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

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.España. Ministerio de Economía y Competitividad AGL2014-54413-REspaña Junta de Andalucia, Consejería de Economía, Innovación y Ciencia BIO-163España Junta de Andalucia, Consejería de Economía, Innovación y Ciencia P10-CVI-636

Genetics of symbiosis in Lotus japonicus: Recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci

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.European Union HPRN-CT-2000-00086, MRTN-CT-2003-505227National Sciences and Engineering Research Council 3277A01Ministerio de Educación y Ciencia BFU2005-0312

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.España FEDER-Ministerio de Economía y Competitividad AGL2014-54413-REspaña Junta de Andalucía,Consejería de Economía, Innovación y Ciencia, P1O-CVI-6368España Junta de Andalucía,Consejería de Economía, Innovación y Ciencia, BIO-16

Lack of validation of genetic variants associated with anti-tumor necrosis factor therapy response in rheumatoid arthritis: a genome-wide association study replication and meta-analysis

Introduction: In this study, our aim was to elucidate the role of four polymorphisms identified in a prior large genome-wide association study (GWAS) in which the investigators analyzed the responses of patients with rheumatoid arthritis (RA) to treatment with tumor necrosis factor inhibitors (TNFi). The authors of that study reported that the four genetic variants were significantly associated. However, none of the associations reached GWAS significance, and two subsequent studies failed to replicate these associations. Methods: The four polymorphisms (rs12081765, rs1532269, rs17301249 and rs7305646) were genotyped in a total of 634 TNFi-treated RA patients of Spanish Caucasian origin. Four outcomes were evaluated: changes in the Disease Activity Score in 28 joints (DAS28) after 6 and 12 months of treatment and classification according to the European League Against Rheumatism (EULAR) response criteria at the same time points. Association with DAS28 changes was assessed by linear regression using an additive genetic model. Contingency tables of genotype and allele frequencies between EULAR responder and nonresponder patients were compared. In addition, we combined our data with those of previously reported studies in a meta-analysis including 2,998 RA patients. Results: None of the four genetic variants showed an association with response to TNFi in any of the four outcomes analyzed in our Spanish patients. In addition, only rs1532269 yielded a suggestive association (P = 0.0033) with the response to TNFi when available data from previous studies were combined in the meta-analysis. Conclusion: Our data suggest that the rs12081765, rs1532269, rs17301249 and rs7305646 genetic variants do not have a role as genetic predictors of TNFi treatment outcomes

Biosíntesis de Glutamato en "Chlamydomonas reinhardii" purificación y propiedades de las encimas con actividad Glutamato Sintasa

De forma resumida, los puntos más importantes que aporta este trabajo son los siguientes: 1. Puesta a punto t caracterización de un ensayo adecuado para medir la actividad GOGAT en Chlamydomonas reinhardii, tanto in situ como in vitro. 2. Estudios in vivo de asimilación de amonio, utilizando distintas estirpes de Chlamydomonas reinhardii, y efecto de distintas condiciones metabólicas sobre el nivel de las actividades NADH- y Fd-GOGAT. 3. Purificación parcial de la NADH-GOGAT y estudio físico-químico y cinético de la proteína. 4. Purificación hasta homogeneidad, caracterización fisicoquímica y cinética, y estudio de la composición de grupos prostéticos de las proteínas del sistema ferredoxina/Fd-GOGAT

La comunicación en la pareja: comunicación en superficie - comunicación en profundidad

Club AntaresClub UNESCO SevillaConsejería de CulturaConvenio Universidad de Sevilla - Fundación El MonteDiputación de Sevill

Flavonoids and isoflavonoids biosynthesis in the model legume lotus japonicus; connections to nitrogen metabolism and photorespiration

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.Ministerio de Ciencia, Innovación y Universidades (MCIU) RTI2018-093571-B-100España Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER, EU) Project US-1256179España Junta de Andalucía, FEDER and Universidad de Sevilla project AGL2014-54413-