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

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

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

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

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

On the Relationship between Corneal Biomechanics, Macrostructure, and Optical Properties

Optical properties of the cornea are responsible for correct vision; the ultrastructure allows optical transparency, and the biomechanical properties govern the shape, elasticity, or stiffness of the cornea, affecting ocular integrity and intraocular pressure. Therefore, the optical aberrations, corneal transparency, structure, and biomechanics play a fundamental role in the optical quality of human vision, ocular health, and refractive surgery outcomes. However, the inter-relationships of those properties are not yet reported at a macroscopic scale within the hierarchical structure of the cornea. This work explores the relationships between the biomechanics, structure, and optical properties (corneal aberrations and optical density) at a macro-structural level of the cornea through dual Placido-Scheimpflug imaging and air-puff tonometry systems in a healthy young adult population. Results showed correlation between optical transparency, corneal macrostructure, and biomechanics, whereas corneal aberrations and in particular spherical terms remained independent. A compensation mechanism for the spherical aberration is proposed through corneal shape and biomechanics

In vivo biomechanical response of the human cornea to acoustic waves

The cornea is the optical window to the brain. Its optical and structural properties are responsible for optical transparency and vision. The shape, elasticity, rigidity, or stiffness are due to its biomechanical properties, whose stability results in ocular integrity and intraocular pressure dynamics. Here, we report in vivo observations of shape changes and biomechanical alterations in the human cornea induced by acoustic wave pressure within the frequency range of 50–350 Hz and the sound pressure level of 90 dB. The central corneal thickness (CCT) and eccentricity (e2) were measured using Scheimpflug imaging and biomechanical properties [corneal hysteresis (CH) and intraocular pressure (IOP)] were assessed with air-puff tonometry in six young, healthy volunteers. At the specific 150 Hz acoustic frequency, the variations in e2 and CCT were 0.058 and 7.33 µm, respectively. Biomechanical alterations were also observed in both the IOP (a decrease of 3.60 mmHg) and CH (an increase of 0.40 mmHg)

Arginine biosynthesis and utilization in maritime pine

Vegetative propagation through somatic embryogenesis in combination with the cryopreservation of embryogenic lines is a major tool in conifer biotechnology. An important process during the maturation phase of embryogenesis is the biosynthesis and deposition of storage proteins. The accumulation of some abundant storage proteins in maturing cotyledonary somatic embryos (SE) is much lower than in mature zygotic embryos (ZE) showing an important influence of storage compounds on the quality of SE. Arginine constitutes a large portion of the amino acid pool in storage proteins of conifers and therefore arginine biosynthesis and utiization is a relevant metabolic pathway during pine embryogenesis and early growth. Research in our laboratory is focused on maritime pine (Pinus pinaster Ait.), a broadly planted conifer species in France, Spain and Portugal where it is distributed over approximately 4 million hectares. This conifer species is also one of the most advanced model trees for genetic and phenotypic studies and a large number of molecular and transcriptomic resources are currently available. With the aim to understand the molecular basis of the differential accumulation of storage proteins in SE and ZE, the arginine metabolic pathway has been studied in maritime pine, in collaboration with the French private institute FCBA. A general overview of this research programme will be presented and discussed. The knowledge acquired from our studies will help to refine biotechnological procedures for clonal propagation of conifers via somatic embryogenesis. Funding support by:The Spanish Ministerio de Economía y Competitividad (BIO2015-69285-R) and Junta de Andalucía (BIO-474). And the French Ministry of Agriculture (DGAL, N°2014-352, QuaSeGraine project). The project also benefited from the technical support of the XYLOBIOTECH facility (ANR-10-EQPX-16 XYLOFOREST).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