Genómica funcional del metabolismo del nitrógeno en relación con la fotorrespiración y estrés abiótico en plantas de Lotus Japonicus

Tesis descargada de TESEOLa asimilación de nitrógeno y el estrés abiótico, particularmente la sequía, son dos de los factores que más limitan el crecimiento de las plantas en el género Lotus. De ahí que la disponibilidad de nitrógeno sea de vital importancia para la supervivencia de las mismas y que éstas hayan desarrollado diferentes mecanismos de regulación metabólicos para garantizar el mejor aprovechamiento de este elemento. La utilización de nitrógeno por las plantas implica varias etapas, que incluyen el transporte de las diferentes fuentes nitrogenadas desde el exterior al interior celular; su posterior reducción y asimilación dentro de las células (asimilación primaria), y, también, la translocación o removilización (reciclaje del nitrógeno; asimilación secundaria) en el interior del vegetal. Estudios previos han demostrado la importancia de una de las rutas implicadas en la asimilación secundaria de nitrógeno, la fotorrespiración. Basándose en cálculos aproximados de la fotorrespiración en hojas de trigo, se determinó que la tasa de nitrógeno asimilado en dicha ruta, en forma de amonio, podría sobrepasar en 10 veces la tasa calculada de asimilación primaria nitrógeno. En esta Tesis Doctoral se ha abordado el estudio, desde el punto de vista de la genómica funcional, del metabolismo del nitrógeno y su relación con la fotorrespiración y con el estrés abiótico en la leguminosa modelo Lotus japonicus. La disponibilidad de mutantes Ljgln2-2, carentes por completo de actividad y proteína glutamina sintetasa plastídica, ha permitido avanzar en el esclarecimiento de la significación fisiológica exacta de dicha enzima en relación con la fotorrespiración y el estrés abiótico, haciendo uso de datos transcriptómicos y metabolómicos y análisis de la co-expresión de redes de genes. Entre las conclusiones más destacadas se incluyen la descripción de la existencia de una regulación coordinada de los genes fotorrespiratorios bajo condiciones de alta acumulación de NH4+; la inducción de rutas alternativas al ciclo GS2 / Fd-GOGAT para la reasimilación del NH4+; la interconexión de la fotorrespiración con el metabolismo central del carbono, del nitrógeno y metabolismo secundario; y el papel de la glutamina sintetasa plastídica en la respuesta al balance C / N y su relación con el metabolismo de la prolina y el estrés hídrico

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

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

A blood microRNA classifier for the prediction of ICU mortality in COVID-19 patients: a multicenter validation study

Background: The identification of critically ill COVID-19 patients at risk of fatal outcomes remains a challenge. Here, we first validated candidate microRNAs (miRNAs) as biomarkers for clinical decision-making in critically ill patients. Second, we constructed a blood miRNA classifier for the early prediction of adverse outcomes in the ICU. Methods: This was a multicenter, observational and retrospective/prospective study including 503 critically ill patients admitted to the ICU from 19 hospitals. qPCR assays were performed in plasma samples collected within the first 48 h upon admission. A 16-miRNA panel was designed based on recently published data from our group. Results: Nine miRNAs were validated as biomarkers of all-cause in-ICU mortality in the independent cohort of critically ill patients (FDR < 0.05). Cox regression analysis revealed that low expression levels of eight miRNAs were associated with a higher risk of death (HR from 1.56 to 2.61). LASSO regression for variable selection was used to construct a miRNA classifier. A 4-blood miRNA signature composed of miR-16-5p, miR-192-5p, miR-323a-3p and miR-451a predicts the risk of all-cause in-ICU mortality (HR 2.5). Kaplan‒Meier analysis confirmed these findings. The miRNA signature provides a significant increase in the prognostic capacity of conventional scores, APACHE-II (C-index 0.71, DeLong test p-value 0.055) and SOFA (C-index 0.67, DeLong test p-value 0.001), and a risk model based on clinical predictors (C-index 0.74, DeLong test-p-value 0.035). For 28-day and 90-day mortality, the classifier also improved the prognostic value of APACHE-II, SOFA and the clinical model. The association between the classifier and mortality persisted even after multivariable adjustment. The functional analysis reported biological pathways involved in SARS-CoV infection and inflammatory, fibrotic and transcriptional pathways. Conclusions: A blood miRNA classifier improves the early prediction of fatal outcomes in critically ill COVID-19 patients.11 página

Manual de Tutoría y Orientación Educativa

Material destinado a los tutores y tutoras de los niveles de primaria y secundaria. En este material, se presenta el marco conceptual que sustenta la tutoría y orientación educativa, así como un conjunto de sesiones de tutoría sobre temáticas relacionadas con el bienestar y desarrollo de los y las estudiantes. Estas sesiones buscan ser un punto de partida para que cada tutor y tutora diseñe e implemente otras, que respondan a las particularidades de sus estudiantes, su contexto sociocultural y su etapa de desarrollo

Effect of viral storm in patients admitted to intensive care units with severe COVID-19 in Spain: a multicentre, prospective, cohort study

Background: The contribution of the virus to the pathogenesis of severe COVID-19 is still unclear. We aimed to evaluate associations between viral RNA load in plasma and host response, complications, and deaths in critically ill patients with COVID-19. Methods: We did a prospective cohort study across 23 hospitals in Spain. We included patients aged 18 years or older with laboratory-confirmed SARS-CoV-2 infection who were admitted to an intensive care unit between March 16, 2020, and Feb 27, 2021. RNA of the SARS-CoV-2 nucleocapsid region 1 (N1) was quantified in plasma samples collected from patients in the first 48 h following admission, using digital PCR. Patients were grouped on the basis of N1 quantity: VIR-N1-Zero (2747 N1 copies per mL). The primary outcome was all-cause death within 90 days after admission. We evaluated odds ratios (ORs) for the primary outcome between groups using a logistic regression analysis. Findings: 1068 patients met the inclusion criteria, of whom 117 had insufficient plasma samples and 115 had key information missing. 836 patients were included in the analysis, of whom 403 (48%) were in the VIR-N1-Low group, 283 (34%) were in the VIR-N1-Storm group, and 150 (18%) were in the VIR-N1-Zero group. Overall, patients in the VIR-N1-Storm group had the most severe disease: 266 (94%) of 283 patients received invasive mechanical ventilation (IMV), 116 (41%) developed acute kidney injury, 180 (65%) had secondary infections, and 148 (52%) died within 90 days. Patients in the VIR-N1-Zero group had the least severe disease: 81 (54%) of 150 received IMV, 34 (23%) developed acute kidney injury, 47 (32%) had secondary infections, and 26 (17%) died within 90 days (OR for death 0·30, 95% CI 0·16-0·55; p<0·0001, compared with the VIR-N1-Storm group). 106 (26%) of 403 patients in the VIR-N1-Low group died within 90 days (OR for death 0·39, 95% CI 0·26-0·57; p<0·0001, compared with the VIR-N1-Storm group). Interpretation: The presence of a so-called viral storm is associated with increased all-cause death in patients admitted to the intensive care unit with severe COVID-19. Preventing this viral storm could help to reduce poor outcomes. Viral storm could be an enrichment marker for treatment with antivirals or purification devices to remove viral components from the blood.This work was supported by grants from the Instituto de Salud Carlos III (FONDO-COVID19, COV20/00110, CIBERES, 06/06/0028; AT), Proyectos de Investigación en Salud (PI19/00590; JFB-M), Miguel Servet (CP20/00041; DdG-C), Sara Borrell (CD018/0123; APT), and Predoctorales de Formación en Investigación en Salud (FI20/00278; AdF). We also received funds from Programa de Donaciones Estar Preparados, UNESPA (Madrid, Spain), and from the Canadian Institutes of Health Research (CIHR OV2–170357; DJK and JFB-M), Research Nova Scotia, Li-Ka Shing Foundation (DJK), and finally by a Research Grant 2020 from ESCMID (APT). COV20/00110, PI19/00590, CP20/00041, CD018/0123, FI20/00278 were co-funded by European Regional Development Fund and European Social Fund (A way to make Europe, and Investing in your future). We thank the IRB-Lleida Biobank 119 (B.0000682) and Plataforma Biobancos PT17/0015/0027 in Lleida, the Hospital Clinic Barcelona (HCB)-IDIBAPS biobank in Barcelona, and the National DNA Bank and the Hospital Universitario de Salamanca biobank (both in Salamanca) for their logistical support with sample processing and storage. We are indebted to the Fundació Glòria Soler for its contribution and support to the COVIDBANK of HCBIDIBAPS Biobank. This work was not supported by any pharmaceutical company or other agency.S

Cualificación en los Objetivos establecidos en la Agenda 2030 de estudiantes y profesores en el Máster Universitario en Profesor de Educación Secundaria Obligatoria y Bachillerato, Formación Profesional y Enseñanza de Idiomas (MUPES)

Memoria ID2022-157 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2022-2023