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

Identificación y regulación transcripcional de genes arogenato deshidratasa implicados en la biosíntesis de kignina en pino marítimo

La fenilalanina es un aminoácido esencial para la síntesis de proteínas pero también un precursor de una gran variedad de compuestos del metabolismo secundario que son esenciales para el crecimiento, desarrollo y defensa de las plantas. La biosíntesis de la pared celular secundaria que tiene lugar durante la formación de madera en árboles implica la biosíntesis masiva de lignina, un polímero que no contiene nitrógeno pero que deriva metabólicamente de la fenilalanina. Por lo tanto, estas plantas requieren una coordinación metabólica precisa entre la biosíntesis de fenilalanina y la biosíntesis de lignina para asegurar su desarrollo y crecimiento anual. En este estudio, hemos encontrado que la enzima arogenato deshidratasa, que cataliza el último paso en la ruta biosintética de la fenilalanina en las plantas, se regula transcripcionalmente a través de la interacción directa con el factor de transcripción PpMYB8. El análisis transcripcional de plantas de pino marítimo silenciadas para PpMyb8 sugiere que este factor de transcripción está directamente involucrado en la biogénesis de la pared celular secundaria y en los procesos de muerte celular. En conjunto, estos resultados indican que un único factor de transcripción coordina la biosíntesis de fenilalanina y la acumulación de lignina durante la formación de madera en las coníferas.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

El análisis transcriptómico de embriones somáticos y cigóticos de pino revela diferencias de expresión en diferentes rutas metabólicas

La embriogénesis es un proceso complejo en las plantas, pero comprenderlo en las coníferas es especialmente crítico no sólo para poder hacer estudios comparativos con angiospermas sino también para producir embriones viables y de calidad con diferentes propósitos. Actualmente falta una visión comparativa global de los genes implicados en la embriogénesis somática y cigótica del pino. En este trabajo presentamos un análisis del transcriptoma en tres etapas del desarrollo de los embriones, identificando procesos biológicos conservados y funciones genéticas activas durante el proceso de embriogénesis somática y cigótica. La mayoria de las diferencias son más significativas a medida que avanza el desarrollo. Cuando comparamos etapas de desasrrollo similares, como es el estadio embrión maduro encontramos 1640 genes sobreexpresados en embriones cigóticos frente a 4814 genes en embriones somáticos.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

Background: There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (~23.8 Gb/C). [br/] Methodology/Principal Findings: A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). [br/] Conclusions/Significance: This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome

Diversification of Fungal Specific Class A Glutathione Transferases in Saprotrophic Fungi

Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes and endogenous metabolism. The distribution of fungal-specific class A GSTs was investigated in saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight GSTFuA isoforms from Phanerochaete chrysosporium and Coprinus cinereus demonstrated functional diversity in saprotrophic fungi. The three-dimensional structures of three P. chrysosporium isoforms feature structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs function as ligandins with various small aromatic compounds, derived from lignin degradation or not, at a L-site overlapping the glutathione binding pocket. By combining genomic data with structural and biochemical determinations, we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry

Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology

The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including beta-etherase that specifically cleaves the beta-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular beta-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. (c) 2012 Elsevier B.V. All rights reserved