Analysis of NPF and NRT transporter families regarding the nitrate nutrition in maritime pine (Pinus pinaster)

Nitrogen is an essential element for life and the main limiting nutrient for plant growth and development1. The main forms of inorganic nitrogen in soils are nitrate and ammonium, which relative abundances depend on environmental conditions such as temperature. In agricultural soils the most abundant nitrogen form is nitrate because the use of chemical fertilizers however in natural ecosystems nitrogen soil composition can be more complex. Conifers are tree gymnosperms with a wide distribution although their large forests dominate the boreal ecosystems where nitrification is limited and ammonium is the main nitrogen soil source2. In this context, conifers have an appreciable tolerance to ammonium. Maritime pine (Pinus pinaster Aiton) is a conifer from the western Mediterranean region of high economic and ecological interest in Spain, France and Portugal. This pine is also a research model tree with different genomic resources such as a reference transcriptome and a gene expression atlas3. Taking advantage of these resources the members of the NPF and NRT transporter families involved in nitrate uptake and transport have been identified and analyzed in maritime pine4. Among the transporter families, the NRT3 one is expanded and composed by six members. The capacity of maritime pine to use nitrate or ammonium has been analyzed in seedlings. The development and growth responses to nitrate nutrition are comparable to ammonium supply. At molecular level, there are strong gene expressions for genes involved in nitrate uptake and assimilation such as Nitrate Reductase, Nitrite Reductase, Glutamine Synthetase 1a, three NRT3 genes and different NPF family members in the different organs. Since the NPF proteins can transport different metabolites, peptides and hormones, the NPF transporters involved in nitrate transport are being identified.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This project was supported by the grant MicroNUpE, BIO2015-73512-JIN; MINECO/AEI/FEDER, UE. JMVM was supported by a grant from the Spanish Ministerio de Educación y Formación Profesional (FPU17/03517) and FO by a grant from the Universidad de Málaga (Programa Operativo de Empleo Juvenil vía SNJG, UMAJI11, FEDER, FSE, Junta de Andalucía)

An unexpected actor in ammonium assimilation in conifer trees

Conifers are tree species with enormous environmental and economic interests but with several characteristics that complicate their investigation (big size, secondary compounds, large long-life cycles, megagenomes…). However, they are well adapted to ammonium-rich soils being a good model to study ammonium assimilation in plants. Although they have a special feature, only two glutamine synthetase (GS, EC 6.3.1.2) genes, GS1a and GS1b, coding for cytosolic proteins, have been identified. In angiosperms and in the gymnosperm Ginkgo biloba there are two types of this enzyme responsible of the ammonium assimilation: GS1 expressed in the cytosol and GS2 in the plastids. Until the date, the searches of new GS1 and GS2 genes in conifers have been made with classical biochemical and molecular biology techniques without satisfactory results. In the present context, the emergence of the next generation sequencing (NGS) techniques has open new opportunities in the resolution of old problems. They have allowed the whole sequencing of the massive conifer genomes and the analysis of their transcriptomes. Thus, in the framework of the European project ProCoGen, a gene expression atlas of the tissues of one-month seedlings was carried out using laser capture microdissection (LCM) and massive sequencing in maritime pine (Pinus pinaster), which is a conifer tree from the Southwestern Mediterranean region1. From the analysis of this work, a new gene coding for a new putative cytosolic GS has been identified, PpGS1c. 1Cañas, RA et al. (2017). Plant J, 91. 1064-1087Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Project funding by Ministerio de Economía y Competitividad BIO2015-69285-R and MicroNUpE (BIO2015-73512-JIN; MINECO/AEI/FEDER, UE

Influencia del envejecimiento del preg-preg en las propiedades de los preg-preg y laminados

IV CONGRESO NACIONAL DE MATERIALES COMPUESTOS. Celebrado en Gijón, los días 21,22 y 23 de Noviembre de 2001Uno de los ensayos más empleados para recalificar prepregs es el test de calorimetría diferencial de barrido D.S.C.. Dicho test permite evaluar cómo la matriz polimérica se ha podido degradar durante el almacenamiento, dado que el objeto del test es determinar los parámetros que controlan la reacción de curado de la resina. Uno de los objetivos de este trabajo es estudiar el efecto que el envejecimiento de la resina tiene sobre los parámetros que controlan el proceso de curado y como una alteración de estos puede o no afectar a las propiedades mecánicas de los laminados.D.S.C. is one of the most used tests to recalify prepregs. This kind of test allows to evaluate how the polymeric matrix has been degraded during the storage due to the fact that the aim of the test is to determine the parameters that control the curing reaction of the resin. One of the objectives of this article is to study the effect of resin ageing on the parameters that controls the curing cycle and how a variation of these can affect the mechanical properties of the laminates.Unión Europea FD97-058

Comparing Bayesian and Montecarlo localization for a robot with local vision

Position estimation is one of the classic problems in mobile robotics. The goal of this paper is to compare two probabilistic localization methods based on local vision for a mobile robot. The experimental set up is based on the Aibo league of the RoboCup, where the robotic dogs major sensor is the on-board camera. Two localization algorithms, Bayesian and Montecarlo (MCL), have been implemented and compared, and their behaviour studied in several situations using a simulato

Transcriptomics of ammonium nutrition in the conifer Pinus pinaster Aiton

Nitrogen is an important element for all living beings because it is part of macromolecules as significant as nucleic acids or amino acids. For plants, it constitutes a limiting factor in their growth and development1 due to their low natural availability in soils thus limiting primary production in ecosystems2. Conifers are a group of gymnosperm plants that form large forest extensions of vegetation, being the main constituents of forests in boreal ecosystems3 where ammonium is the main source of inorganic nitrogen4. Due to the characteristics of the soils in which conifers usually grow, these plants have developed a high tolerance to the presence of ammonium, which may constitute their main source of inorganic nitrogen5. The maritime pine (Pinus pinaster Aiton) is a conifer that has a wide distribution in the western Mediterranean area and has been widely used in reforestation, soil stabilization tasks and industrially. In recent years, maritime pine has been the subject of multiple omic studies that have resulted in the acquisition of important tools and resources6,7. The present work is focused on the analysis of the ammonium uptake and management efficiency, and its relationship with the biomass accumulation in maritime pine. For this purpose, several experiments have been developed in which pine seedlings have undergone different levels of ammonium nutrition, both in the short and long term. As a result of short-term experiments, the characterization of transcriptomic response to the process of ammonium nutrition (uptake and assimilation) is being studied at mRNA, lncRNA and miRNA level in roots. In relation to long-term experiments, ten different provenances of maritime pine seedlings were treated with different ammonium levels and the biomass changes were measured. The results obtained suggest the existence a certain phenotypic plasticity grade for this conifer.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This project was supported by a grant form the Spanish Ministerio de Ciencia, Innovación y Universidades (MicroNUpE, BIO2015-73512-JIN; MINECO/AEI/FEDER, UE). FO was supported by a grant from the Universidad de Málaga (Programa Operativo de Empleo Juvenil vía SNJG, UMAJI11, FEDER, FSE, Junta de Andalucía) and JMVM by a grant from the Spanish Ministerio de Educación y Formación Profesional (FPU17/03517

Involvement of miRNAs in the short-term response of pine roots to ammonium nutrition

Post-transcriptonal gene regulation is an essential control point regarding gene expression programs. A class of small non-coding RNAs, microRNAs (miRNAs) play important roles during this process. They act on target mRNAs through post-transcriptional gene silencing, either by endonuclease mediated transcript cleavage or by translational repression of targeted mRNAs (Pattanayak et al., 2013). Thus, miRNAs are involved in the regulation mechanisms of important plant processes, including the regulation of the processes related to nitrogen nutrition (Gutiérrez, 2012). In the present work, maritime pine (Pinus pinaster Aiton) was used to study the role of miRNAs regarding NH4+ nutrition in conifers, a group of plants that exhibits tolerance to NH4+ nutrition compared to NO3- nutrition specially during seedling stage Ortigosa et al., 2022). This fact is of high interest since numerous NH4+ nutrition can negatively affect the growth and development of different crops. The global miRNA expression has been characterized in the roots of maritime pine seedlings after 2 h and 24 h from fertilization with two levels of NH4+ solutions (0.1 mM and 3 mM). The miRNA-seq analysis revealed 271 new miRNAs with an identified precursor although only 4 were differentially expressed. Gutiérrez RA. 2012. Science, 336:1673-1675. Ortigosa F, et al. 2022. Plant Cell and Environment, 45,915-935. Pattanayak D, et al. 2013. Plant Molecular Biology Reporter, 31, 493-506.Acknowledgements & Funding. This work was supported by the grants BIO2015-73512-JIN MINECO/AEI/FEDER, UE; P20_00036 PAIDI 2020/FEDER, UE, and B4-2021-01 (Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech). JMVM was supported by the grant FPU17/03517

Exosomes: A key piece in asthmatic inflammation

Asthma is a chronic disease of the airways that has an important inflammatory compo-nent. Multiple cells are implicated in asthma pathogenesis (lymphocytes, eosinophils, mast cells, basophils, neutrophils), releasing a wide variety of cytokines. These cells can exert their inflammatory functions throughout extracellular vesicles (EVs), which are small vesicles released by do-nor cells into the extracellular microenvironment that can be taken up by recipient cells. Depending on their size, EVs can be classified as microvesicles, exosomes, or apoptotic bodies. EVs are heterogeneous spherical structures secreted by almost all cell types. One of their main functions is to act as transporters of a wide range of molecules, such as proteins, lipids, and microRNAs (miRNAs), which are single-stranded RNAs of approximately 22 nucleotides in length. Therefore, exosomes could influence several physiological and pathological processes, including those involved in asthma. They can be detected in multiple cell types and biofluids, providing a wealth of infor-mation about the processes that take account in a pathological scenario. This review thus summarizes the most recent insights concerning the role of exosomes from different sources (several cell populations and biofluids) in one of the most prevalent respiratory diseases, asthma.This manuscript was supported by Fondo de Investigación Sanitaria–FIS and FEDER (Fondo Europeo de Desarrollo Regional) [PI15/00803, PI18/00044, and FI16/00036], CIBERES, Merck Health Foundation funds, and RTC-2017-6501-1 (Ministerio de Ciencia, Innovación y Universidades)

Nuevas aproximaciones a la asimilación del amonio en la conífera Pinus pinaster

Comunicación oralLas coníferas son plantas bien adaptadas a crecer en suelos con considerables cantidades de amonio. Por este motivo son un buen modelo de estudio de la asimilación de amonio en plantas. El pino marítimo (Pinus pinaster Aiton) es una especie de conífera autóctona del Mediterráneo occidental con gran importancia medio ambiental y económica. En el marco del proyecto europeo ProCoGen se realizó un atlas trasncriptómico de los tejidos de plántulas de un mes de pino marítimo mediante el uso de microdisección por captura con láser (LCM)1. El análisis de este atlas de expresión génica ha permitido la caracterización de los genes implicados en metabolismo nitrogenado en plántulas de pino marítimo y la identificación de nuevos genes con expresión baja o muy localizada. Entre estos se encuentra un gen codificante para una glutamina sintetasa citosólica no conocida hasta el momento (PpGS1c). Cuando se realizan búsquedas tanto en la base de datos transcriptómica de pino marítimo como en los ensamblajes de los genomas de diferentes coníferas no se identifican GS de tipo plastidial pero sí las respectivas GS1c al igual que el resto de GS citosólicas de coníferas (GS1a y GS1b). La GS1c en pino marítimo presenta una expresión especialmente baja y muy localizada tanto en el meristemo apical como en el meristemo radicular. Al estudiar su promotor se encuentran posibles sitios de unión de factores de transcripción reguladores de la expresión génica en zonas en desarrollo. En paralelo al estudio de este gen se está desarrollando un estudio transcriptómico para identificar y caracterizar funcionalmente los genes implicados en la absorción y asimilación del amonio, y en la regulación de ambos procesos. Para ello se están realizando experimentos de nutrición amoniacal en los que se obtienen muestras de diferentes tejidos de la raíz utilizando la técnica de LCM.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Financiación: Proyectos del Ministerio de Economía y Competitividad BIO2015-69285-R y MicroNUpE (BIO2015-73512-JIN; MINECO/AEI/FEDER, UE)

Análisis transcriptómico de la eficiencia en la captación de nitrógeno y la producción de biomasa en pino marítimo (Pinus pinaster)

PanelLas coníferas son un grupo de plantas gimnospermas que forman extensos bosques, principalmente en el hemisferio norte1, por lo que presentan importantes contribuciones ecológicas y económicas. Debido al tipo de suelos en los que suelen crecer las coníferas, estas plantas han desarrollado una gran tolerancia al amonio, muchas coníferas son capaces de usarlo como su principal fuente de nitrógeno inorgánico2. El pino marítimo (Pinus pinaster Aiton) es una conífera distribuida por el Mediterráneo occidental que ha sido utilizada en tareas de reforestación, estabilización de suelos e industrialmente para la obtención de madera y resina. En los últimos años, esta conífera ha sido objeto de múltiples estudios genéticos y ómicos, obteniéndose importantes herramientas para su estudio3. En el presente trabajo se pretende analizar la eficiencia en la captación de amonio y su relación con la producción de biomasa. Para ello se están desarrollando experimentos en los que se someten a plántulas de pino marítimo a diferentes niveles de nutrición amoniacal tanto a corto como a largo plazo. A partir de estos experimentos se quiere estudiar la respuesta génica de estas plantas a la nutrición amoniacal, la captación, su asimilación y la regulación de ambos procesos. El proyecto se centra en la respuesta que presenta la zona de desarrollo de la raíz, diferenciando los distintos tejidos que se pueden encontrar en ella mediante el uso de la técnica de microdisección por captura láser (LCM). Finalmente, el transcriptoma de estas muestras será analizado mediante RNA-Seq.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.Financiación: Proyectos del Ministerio de Economía y Competitividad BIO2015-69285-R y MicroNUpE (BIO2015-73512-JIN; MINECO/AEI/FEDER, UE). Francisco Ortigosa ha realizado el trabajo contratado por el Programa Operativo de Empleo Juvenil vía SNJG (UMAJI11)

Study of the NPF and NRT transporter families in the conifer Pinus pinaster

Nitrogen, in natural environments, is the main limiting nutrient for plant growth and development1. This essential element can be presented in different forms in the soils being the two main forms nitrate and ammonium, although the relative abundance of these compounds depends on environmental conditions. In plants, nitrate uptake and transport involve the function of specific transporters, mainly of the NRT and NPF families. Genes encoding these transporters have been well studied in crop plants since in agricultural soils nitrate has become the predominant nitrogen compound as a result of the intensive use of fertilizers2. However, in soils of the large conifer forests dominating the boreal ecosystems, ammonium is the main source of inorganic nitrogen due to the limited nitrification process3. Consequently, conifers have been told to have a preference for ammonium over nitrate and only limited information is available about nitrate transporters in these gymnosperms. Maritime pine (Pinus pinaster Aiton) is a conifer tree with a wide distribution in the western Mediterranean region and with a great morphological and physiological plasticity. This pine also has a good resistance to abiotic stress4. In maritime pine, the genomic resources have been improved in the last few years5 allowing the identification and molecular analysis of members of the NRT and NPF gene families6. Growth and development responses have been compared on pine seedlings cultured under different nitrate and ammonium supplies. Gene expression have been analyzed and the results show a strong expression of different genes related with the uptake, transport and assimilation of nitrate in plants such as nitrate and nitrite reductases, glutamine synthetase and some genes from the NRT and NPF families, suggesting they are involved in nitrate acquisition from soil.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This project was supported by the grant MicroNUpE, BIO2015-73512-JIN; MINECO/AEI/FEDER, UE. JMVM was supported by a grant from the Spanish Ministerio de Educación y Formación Profesional (FPU17/03517) and FO by a grant from the Universidad de Málaga (Programa Operativo de Empleo Juvenil vía SNJG, UMAJI11, FEDER, FSE, Junta de Andalucía)