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

Una nueva capa de información: epitranscriptómica

The nucleosides of nucleic acids (DNA and RNA) undergo chemical modifications, mainly in their nitrogenous bases, which constitute a new layer of information. The set of modifications suffered by RNA is known as epitranscriptome. In this article, we review the physiological importance of the epitranscriptome, mainly in the regulation of translation.Los nucleósidos de los ácidos nucleicos (ADN y ARN) sufren modificaciones químicas, principalmente en sus bases nitrogenadas, que constituyen una nueva capa de información. El conjunto de las modificaciones que sufre el ARN se conoce como epitranscriptoma. En este artículo se hace una revisión de la importancia fisiológica del epitranscriptoma, principalmente en la regulación de la traducción

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)

Transcriptional regulation os phenylalaline biosynthesis and utilization

Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and utilization should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown in conifers that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. Constitutive overexpression of Myb8 in white spruce increased secondary-wall thickening and led to ectopic lignin deposition (Bomal et al. 2008). In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. Functional orthologues of members of this network described have been identified in poplar and eucalyptus, but in conifers functional evidence had only been obtained for MYBs. We have identified in the P. pinaster genome 37 genes encoding NAC proteins, which 3 NAC proteins could be potential candidates to be involved in vascular development (Pascual et al. 2015). The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management. This work is supported by the projects BIO2012-33797, BIO2015-69285-R and BIO-474 References: Bomal C, et al. (2008) Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. J Exp Bot. 59: 3925-3939. Craven-Bartle B, et al. (2013) A Myb transcription factor regulates genes of the phenylalanine pathway in maritime pine. Plant J, 74: 755-766. Pascual MB, et al. (2015) The NAC transcription factor family in maritime pine (Pinus pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol, 15: 254.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Métodos de secuenciación: segunda generación

During the Human Genome Project, many technical advances were introduced to the Sanger sequencing method. Those advances allowed the parallelization and the automation of this method, acting as a prelude  for the revolution that leaded to the appearance of a second generation of sequencing methods. With them, it has been achieved a dramatic reduction in the sequencing cost and the production of large amounts of infor- mation from a single nucleic acid sample. This fact has allowed to perform massive studies on genomes and transcriptomes leading to the omics sciences era, in which multiple genomes from different species haven been sequenced, and their functional dynamics have been studied at the genetic, epigenetic and transcriptional level.Durante el desarrollo del Proyecto Genoma Humano fueron introducidos muchos avances técnicos al método de secuenciación de Sanger. Estos permitieron la paralelización y la automatización de este método y sirvieron como preludio de la revolución que supuso la aparición de una segunda generación de métodos de secuenciación. Con ellos se ha conseguido una reducción espectacular de los costes de secuenciación y la producción de grandes cantidades de información a partir de una muestra de ácido nucleico. Esto ha permitido realizar estudios masivos sobre los genomas y los transcriptomas, dando lugar a la era de las ciencias ómicas, durante la cual se han secuenciado múltiples genomas de diferentes especies y se ha estudiado su dinámica funcional a nivel genético, epigenético y transcripciona

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

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

La captura por microdisección láser, desvelando los secretos de una célula

Laser microdissection capture is a powerful microscopy technique that allows the isolation and capture of tissues or individual cells from histological sections or cell cultures. Since the beginning of the 20th century, the bases that led to its final development in 1996 by Emmert-Buck and later marketed by Arcturus. This technique, mainly developed as a tool for pathologists, has spread to different branches of medicine and biology and is an established method in molecular biology, neuroscience, developmental biology, cancer research, forensic medicine, proteomics, metabolomics or plant researchLa captura por microdisección láser  es una poderosa técnica de microscopía que permite el aislamiento y captura de tejidos o células individuales a partir de secciones histológicas o cultivos celulares. Desde principios del siglo XX se han ido cimentando lasbases que dieron lugar a su desarrollo defnitivo en 1996 por Emmert-Buck y comercializado posteriormente por Arcturus . Esta técnica, principalmente desarrollada como una herramienta para los patólogos, se ha extendido a diferentes ramas de la medicina y la biología siendo un método establecido en la biología molecular, las neurociencias, la biología del desarrollo, la investigación del cáncer, la medicina forense, la proteómica, la metabolómica o la investigación de planta