La genómica de las plantas

La mejora vegetal es una actividad tan antigua como la propia agricultura. El estudio de las variedades genéticas de la vegetación permite conocer en profundidad sus funciones y mecanismos para localizar los beneficios alimenticios, textiles, cosméticos o combustibles que la naturaleza ha puesto al servicio del ser humano

Molecular and functional approaches to enhance biomass production in forest trees

Forest trees comprise a large group of angiosperm and gymnosperm species that play a crucial role in global carbon fixation, and maintenance of biodiversity. Forest trees are also of great economic importance since they provide a wide range of products of commercial interest, including wood, pulp, biomass and important secondary metabolites. A sustainable management of forest resources is needed to preserve natural forests and to meet the increasing international demands in the production of wood and other forest-derived products. New advances and developments in biotechnology may contribute to accelerate the domestication of important traits for forest productivity. Nitrogen assimilation and recycling play a key role in tree growth and biomass production and we firmly believe that knowledge on nitrogen metabolism will lead to approaches aimed at increasing forest productivity. We are interested in studying nitrogen metabolism and its regulation in maritime pine (Pinus pinaster Aiton), a forest tree species of great economic and ecological importance in the Mediterranean area and a relevant model for conifer genomic research in Europe. An overview of our research programme will be presented and discussed. Research supported by Spanish Ministry of Economy and Competitiveness and Junta de Andalucía (Grants BIO2012-33797, PLE2009-0016 and research group BIO-114).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Nitrogen metabolism and gene expression landscape in maritime pine

This work was supported by the ProCoGen grant (FP7-KBBE-2011-5) and by the MicroNUpE grant (BIO2015-73512-JIN).Maritime pine (Pinus pinaster Aiton) is one the most important conifer species in the southwestern Mediterranean region because of its economic and environmental potential. For this reason, a work program in functional genomics has been developed in the frame of the ProCoGen project. One objective was to complete the knowledge about P. pinaster transcriptome with the tissue-specific localization of the gene expression of the low accumulated transcripts in sharper regions (Cañas et al. 2017). In order to reach these objectives total RNA was obtained from isolated tissues through laser capture microdissection (LCM). Due to the limiting amount from these extracts, the RNA samples were reverse-transcribed and the resultant cDNA amplified using our CRA+ protocol (Cañas et al. 2014). The obtained reads were assembled to improve the previous reference transcriptome. Reads were mapped against this transcriptome and the read accounts analyzed in order to found gene co-expression networks using the WGCNA software. These results have allowed us the characterization of nitrogen metabolism in maritime pine during the seedling stage stablishing relationships between the different components. This include the identification of new genes with low or very localized expression as occurred for the PpGS1c gene encoding a new cytosolic glutamine synthetase. From this starting point, we are developing a new project, MicroNUpE, to identify and study the genes involved in ammonium uptake and regulation in different root tissues that will be isolated through LCM. Cañas et al. (2017). Plant J, doi:10.1111/tpj.13617. Cañas et al. (2014). Tree Physiol, 34:1278-1288.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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)

Biotechnological approaches to increase biomass production in trees

Nutrient use efficiency is one of the factors influencing growth and therefore of high importance for biomass production in trees. Poplar is a model tree widely used for molecular and functional studies and the characterization of transgenic poplars overexpressing structural and regulatory genes involved in glutamine biosynthesis has provided insights on how glutamine metabolism is involved in N economy and biomass production in woody plant models. Numerous studies have shown the relevance of GS isoenzymes in plant development, biomass production, and yield (Cánovas et al. 2006; Castro-Rodríguez et al. 2015). In this communication two examples of functional analysis of plant genes in poplar, and their potential interest for biotechnological approaches are presented (Pascual et al. 2018; Rueda-López et al. 2017). Overexpression of cytosolic NADP+-isocitrate dehydrogenase (ICDH), one of the major enzymes involved in the production of 2-oxoglutarate for amino acid biosynthesis in plants, yields poplar trees with increased growth and enhanced vascular development in young leaves and apical stems. These plants also show an increased expression of genes associated with vascular differentiation and altered amino acids and organic acids content (Pascual et al. 2018). In other study, we observed that overexpression of Dof5, a transcriptional regulator of lignin production and the carbon-nitrogen balance, produced poplar trees with increased growth and biomass production when N availability in the soil is sufficient (Rueda-López et al. 2017). Taken together, these results suggest a close relationship between carbon and nitrogen metabolism and highlights the relevance of glutamine and glutamate biosynthesis in the control of growth and development. Research supported by Spanish Ministry of Economy and Competitiveness and Junta de Andalucía (Grants BIO2015-69285-R, BIO2012-0474 and research group BIO-114).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

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

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

Análisis molecular y funcional de los transportadores de amonio (AMT) en pino marítimo (Pinus pinaster)

En los bosques de coníferas la mayor parte del nitrógeno inorgánico disponible se encuentra en forma de amonio como fuente primaria de nitrógeno asimilable [1]. El amonio se incorpora por las raíces a través de proteínas de transmembrana denominadas transportadores de amonio (AMT). En nuestro laboratorio se ha llevado a cabo estudios de transcriptómica en diferentes órganos de la conífera modelo Pinus pinaster y bajo diferentes condiciones nutricionales con amonio como fuente de nitrógeno [2]. Los resultados obtenidos se han recogido en una base de datos denominada EuroPineDB (http://www.scbi.uma.es/pindb/) en la que se han identificado 5 isogenes de la familia AMT, 3 isoformas perteneciente a la subfamilia 1: PpAMT1;1, PpAMT1;2 y PpAMT1;3 y 2 pertenecientes a la subfamilia 2: PpAMT2;1 y PpAMT2;2. Nuestro grupo ha comenzado la caracterización molecular de estos genes y se han realizado estudios comparados con angiospermas. Como primera aproximación se han determinando los niveles de expresión y la distribución de transcritos en diferentes órganos de la planta, en diferentes estadios de desarrollo y bajo diferentes condiciones nutricionales. Además, se han realizado construcciones de las correspondientes proteínas recombinantes para estudiar las características bioquímicas de las diferentes isoformas mediante expresión heteróloga en levadura. Los resultados obtenidos indican que los genes AMT de pino se expresan de forma diferencial en distintos órganos de la planta y las proteínas que codifican difieren en sus características moleculares y parámetros cinéticos. En conjunto, nuestros resultados sugieren que los miembros de la familia génica AMT de pino desempeñan funciones diferentes en el transporte de nitrógeno de esta conífera. Bibliografía [1] Cánovas et al. (2007) Journal Experiment Botany 58: 2307–2318. [2] Canales et al. (2010) Amino Acids 39, 4: 991-1001.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Glutamato Sintasas de coníferas: estructura génica y estudios filogenéticos

Las plantas sintetizan glutamato a partir de amonio por la actividad combinada de las enzimas glutamina sintetasa (GS) y glutamato sintasa (GOGAT). En plantas, hay dos formas de glutamato sintasa que difieren en sus donadores de electrones, NADH-GOGAT (EC 1.4.1.14) y Fd-GOGAT (EC 1.4.7.1). Son flavoproteínas complejas de hierro y azufre que contienen dominios involucrados en el control y coordinación de sus actividades catalíticas. En coníferas, se han aislado las secuencias parciales de cDNA para GOGAT y se han usado para estudios de expresión génica. Sin embargo, el conocimiento de la estructura génica y las relaciones filogenéticas con otras enzimas vegetales es bastante escasa. Los avances tecnológicos en la secuenciación de megagenomas de coníferas han permitido obtener las secuencias completas de cDNA que codifican Fd- y NADH-GOGAT de pino marítimo, así como clones BAC que contienen secuencias para genes de NADH-GOGAT y Fd-GOGAT. Hemos estudiamos la organización genómica de los genes GOGAT de pino, el tamaño de sus exones / intrones, número de copias en el genoma y relaciones con otros genes de plantas. Se ha realizado un análisis filogenético, y el estudio del grado de preservación de los dominios clave para la actividad catalítica de estas enzimas en diferentes taxa. Nuestra conclusión es que Fd- y NADH-GOGAT están codificadas por genes de una sola copia en el genoma de pino marítimo. El gen que codifica a Fd-GOGAT es extremadamente grande y abarca más de 330 kb. La presencia de intrones muy largos resalta la importante contribución de los retrotransposones tipo LTR en el tamaño del genoma de las coníferas. Por el contrario la estructura del gen de NADH-GOGAT es similar a la de sus ortólogos en angiospermas. Nuestro análisis filogenético indica que estos dos genes tenían orígenes diferentes durante la evolución de las plantas. Estos resultados proporcionan nuevos conocimientos sobre la estructura y evolución molecular de estos genes esenciales.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The arogenate dehydratase ADT2 is essential for seed development in Arabidopsis

Phenylalanine (Phe) biosynthesis in plants is a key process, as Phe serves as precursor of proteins and phenylpropanoids. The prephenate pathway connects chorismate, final product of the shikimate pathway, with the biosynthesis of Phe and Tyr. Two alternative routes of Phe biosynthesis have been reported: one depending of arogenate, and the other of phenylpyruvate. Whereas the arogenate pathway is considered the main route, the role of the phenylpyruvate pathway remains unclear. Here, we report that the deficiency in ADT2, a bifunctional arogenate dehydratase (ADT)/ prephenate dehydratase (PDT) enzyme, causes embryo arrest and seed abortion. This result makes a clear distinction between the essential role of ADT2 and the five remaining ADTs from Arabidopsis, which display mostly overlapping functions. We have found that PHA2, a monofunctional PDT from yeast, restores the adt2 phenotype when is targeted within the plastids, but not when is expressed in the cytosol. Similar results can be obtained by expressing ADT3, a monofunctional ADT. These results suggest that Phe can be synthesized from phenylpyruvate or arogenate when the bifunctional ADT2 is replaced by other ADT or PDT enzymes during seed formation, highlighting the importance of Phe for embryo development, and providing further insights into the plasticity of Phe biosynthesis.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech