Metabolic channeling of phe for lignin biosynthesis in maritime pine

Phenylalanine (Phe) is the main precursor of phenylpropanoids biosynthesis in plants. This vast family of Phe-derived compounds can represent more than 30% of captured photosynthetic carbon, playing essential roles in plants such as cell wall components, defense molecules, pigments and flavors. In addition to its physiological importance, phenylpropanoids and particularly lignin, a component of wood, are targets in plant biotechnology. The arogenate pathway has been proposed as the main pathway for Phe biosynthesis in plants (Maeda et al., 2010). The final step in Phe biosynthesis, catalyzed by the enzyme arogenate dehydratase (ADT), has been considered as a key regulatory point in Phe biosynthesis, due to its key branch position in the pathway, the multiple isoenzymes identified in plants and the existence of a feedback inhibition mechanism by Phe. So far, the regulatory mechanisms underlying ADT genes expression have been poorly characterized, although a strong regulation of the Phe metabolic flux should be expected depending on its alternative use for protein biosynthesis versus phenylpropanoid biosynthesis. This second fate involves a massive carbon flux compared to the first one. Here we report our current research activities in the transcriptional regulation of ADT genes by MYB transcription factors in Pinus pinaster. The conifers channels massive amounts of photosynthetic carbon for phenylpropanoid biosynthesis during wood formation. We have identified the complete ADT gene family in maritime pine (El-Azaz et al., 2016) and a set of ADT isoforms specifically related with the lignification process. The potential control of transcription factors previously reported as key regulators in pine wood formation (Craven-Bartle et al., 2013) will be presented.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Maritime pine PpMYB8 directly co-regulates secondary cell wall architecture and the associated Phe-biosynthesis pathway

Plants rely on the biosynthesis of L-Phenylalanine as building block for the synthesis of proteins but also as precursor for a tremendous range of plant-derived compounds essential for its grown, development and defense. Polymerization of secondary cell wall in trees involves the massive biosynthesis, among others, of the Phe-derived compound lignin. Thus, these plants require an accurate metabolic coordination between Phe and lignin biosynthesis to ensure its normal development. We have here identified that the pine arogenate dehydratase, whose enzyme activity limits the biosynthesis of Phe in plants, is transcriptionally regulated through direct interaction with PpMyb8. We have also shown that this transcription factor is directly involve in secondary cell wall biogenesis and cell death processes. Together these results indicate that a single transcription factor coordinates lignin accumulation and the proper biosynthesis of its essential precursor L-Phe.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

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene ($\beta$-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods.Transformation génétique du pin maritime (Pinus pinaster Ait.). En appliquant nos protocoles d'embryogenèse somatique développés pour Pinus pinaster, des tissus et plantes transgéniques ont été obtenus après bombardement avec des microparticules (biolistique) ou coculture de masses embryonnaires (ESM) avec Agrobacterium tumefaciens. Les expériences de transformation ont été conduites à l'aide du gène de sélection hpt (résistance à l'hygromycine B) et du gène rapporteur gus (activité $\beta$-glucuronidase). L'hygromycine a permis de sélectionner efficacement les cellules transformées par ces deux méthodes en 4 à 19 semaines. Le nombre moyen de lignées résistantes à l'hygromycine exprimant le gène gus obtenu par gramme d'ESM varie de 7,0 à 8,5 (biolistique) ou de 0 à 67,3 (Agrobacterium). Les embryons matures obtenus à partir de certaines de ces lignées ont pu être convertis en plantules élevées en serre. Seulement 12 mois sont nécessaires de la transformation des ESM jusqu'à l'acclimatation des plantes. La nature transgénique des ESM, embryons somatiques et plantes, a été confirmée à l'aide de tests histochimiques “ GUS " et de méthodes moléculaires

Molecular phenotyping of Maritime pine somatic plants transformed with an RNAi construct targeting cinnamyl alcohol dehydrogenase

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Molecular phenotyping of Maritime pine somatic plants transformed with an RNAi construct targeting cinnamyl alcohol dehydrogenase

We perform large-eddy simulations of neutral atmospheric boundary-layer flow over a cluster of buildings surrounded by relatively flat terrain. The first investigated question is the effect of the level of building detail that can be included in the numerical model, a topic not yet addressed by any previous study. The simplest representation is found to give similar results to more refined representations for the mean flow, but not for turbulence. The wind direction on the other hand is found to be important for both mean and turbulent parameters. As many suburban areas are characterised by the clustering of buildings and homes into small areas separated by surfaces of lower roughness, we look at the adjustment of the atmospheric surface layer as it flows from the smoother terrain to the built-up area. This transition has unexpected impacts on the flow; mainly, a zone of global backscatter (energy transfer from the turbulent eddies to the mean flow) is found at the upstream edge of the built-up area

L'embryogenèse somatique : une méthode de multiplication végétative du pin maritime pour demain ?

FCBA et l’INRA ont engagé des recherches il y a maintenant près de 20 ans pour développer une méthode de multiplication végétative performante du pin maritime. Ses applications dans le programme d’amélioration génétique seraient multiples, depuis la gestion durable des ressources génétiques jusqu’à la sélection plus efficace des meilleures variétés et leur déploiement facilité dans les plantations. Suite aux développements pionniers chez l’épicéa auxquels FCBA a contribué (années 80, Afocel) et comme chez la plupart des autres conifères, c’est le processus d’embryogenèse somatique à partir de graines immatures couplé à la cryoconservation des embryons somatiques obtenus qui offre actuellement les meilleures perspectives pratiques. Des progrès importants ont été obtenus pour la maîtrise de cette technologie, particulièrement depuis la mise en place en 2004 (et continue depuis) d’une collaboration spécifique sur ce thème entre les équipes « Biotechnologie & Sylviculture Avancée » de FCBA et «Amélioration, Génétique et Physiologie Forestières » de l’INRA. Nous faisons ici un bilan des avancées majeures à différentes étapes de l’embryogenèse somatique jusqu’à la mise en place d’essais au champ en cours d’évaluation. Nous portons également un regard sur les verrous techniques et les contraintes socio-économiques qu’il faudrait lever pour amener cette technologie prometteuse à franchir les portes de l’application pratique

Towards functional genomics of transcription factor genes associated to growth and wood formation in maritime pine

Maritime pine is a major forest tree in southern Europe for sustainable delivery of bio-based products through advanced plantation forestry of improved varieties. Early selection is needed to reduce breeding cycle and accelerate variety deployment in the context of climate change. Both gene(s) and genome-wide information should provide opportunities for predictive, marker-assisted selection. Reverse genetics, defined as ectopic expression or silencing of candidate genes can be useful for functional dissection of traits of interest. Functional genomics of 9 transcription factor (TF) genes associated to growth and wood formation (MYB1,8,14,20,23, DOF5, MADBOX4, NACx, NACataf) was initiated in maritime pine through genetic transformation of embryogenic tissue. Twelve TF constructs designed for constitutive overexpression (OE) or silencing (RNAi) were studied: 6 constructs (batch 1) from previous projects (MYB1-RNAi, MYB8-OE/RNAi, MYB14-RNAi, DOF5-OE/RNAi) as well as 3 constructs for putative gene targets of MYB (CAD-RNAi) and DOF5 (GS1a-OE, GS2-OE); and 6 constructs (batch 2) obtained during ProCoGen (MYB23-OE, MYB20-OE, MADBOX4-OE, NACx-OE/RNAi, NACataf-OE). Phosphinothricin-resistant lines could be cryopreserved for all but one (MYB20-OE) constructs. Transformation rate was estimated in the range 9.6-22.0 (OE) or 0.4-18.0 (RNAi) transgenic lines per gram embryogenic tissue (batch 1). Somatic embryos were obtained from 1-3 lines per construct (25 lines in total) and successfully converted to plants (batch 1). Acclimatization rates were similar to controls and transgenic plants were confirmed for most lines. Putative adverse effect of MYB14-RNAi, DOF5-RNAi, and MYB8-OE on transformation rate was observed. DOF5-RNAi apparently stimulated germination rate. Plant growth data and morphology are available for constructs from batch 1 after up to 12 (MYB1-RNAi, MYB8-OE/RNAi, DOF5-OE/RNAi, GS2-OE, GS1a-OE) or 42 months (MYB14-RNAi, CAD-RNAi) growth. Both transgene copy number and targeted gene expression data are available from transgenic plants obtained from MYB14-RNAi and CAD-RNAi lines. Transgenics and controls from batch 1 were sampled at age 16 (MYB1-RNAi, MYB8-OE/RNAi, DOF5-OE/RNAi) or 70 months (MYB14-RNAi, CAD-RNAi). Molecular characterization of MYB1,8,14 and DOF5 plant material has been performed by qPCR and transcriptomic analysis using microarray is underway. Wood analyses have been initiated for MYB14- and CAD-RNAi plants (in progress)