Impaired cell growth under ammonium stress explained by modeling the energy cost of vacuole expansion in tomato leaves

Ammonium (NH4+)-based fertilization efficiently mitigates the adverse effects of nitrogen fertilization on the environment. However, high concentrations of soil NH4+ provoke growth inhibition, partly caused by the reduction of cell enlargement and associated with modifications of cell composition, such as an increase of sugars and a decrease in organic acids. Cell expansion depends largely on the osmotic-driven enlargement of the vacuole. However, the involvement of subcellular compartmentation in the adaptation of plants to ammonium nutrition has received little attention, until now. To investigate this, tomato (Solanum lycopersicum) plants were cultivated under nitrate and ammonium nutrition and the fourth leaf was harvested at seven developmental stages. The vacuolar expansion was monitored and metabolites and inorganic ion contents, together with intracellular pH, were determined. A data-constrained model was constructed to estimate subcellular concentrations of major metabolites and ions. It was first validated at the three latter developmental stages by comparison with subcellular concentrations obtained experimentally using non-aqueous fractionation. Then, the model was used to estimate the subcellular concentrations at the seven developmental stages and the net vacuolar uptake of solutes along the developmental series. Our results showed ammonium nutrition provokes an acidification of the vacuole and a reduction in the flux of solutes into the vacuoles. Overall, analysis of the subcellular compartmentation reveals a mechanism behind leaf growth inhibition under ammonium stress linked to the higher energy cost of vacuole expansion, as a result of alterations in pH, the inhibition of glycolysis routes and the depletion of organic acids.TP benefited from a cotutelle PhD (University of Bordeaux and University of the Basque Country) and thanks the University of the Basque Country (UPV/EHU, Spain) for his PhD grant during the execution of this work. This research was financially supported by the Basque Government (IT-932-16) and the Spanish Government (BIO2017-84035-R co-funded by Fondo Europeo para el Desarrollo Regional [FEDER]). Analytics were supported by MetaboHUB (ANR-11-INBS-0010) and PHENOME (ANR-11-INBS-0012) projects. Technical support was provided by Cedric Cassan, Ana Renovales and Mandy Bordas. The authors also thank SGIker (UPV/EHU, FEDER, EU) for the technical and human support provided

1H-NMR metabolomics: Profiling method for a rapid and efficient screening of transgenic plants

Metabolomics-based approaches are methods of choice for studying changes in fruit composition induced by  environmental or genetic modulation of biochemical pathways in the fruit. Owing to enzyme redundancy and  high plasticity of the metabolic network, transgenic alteration of the activity of the enzymes from the central metabolism very often results in only slight modifications of the fruit composition. In order to avoid costly and  time-consuming plant analysis, we used a fast and sensitive 1H-NMR-based metabolomic profiling technique  allowing discovery of slight metabolite variations in a large number of samples. Here, we describe the  screening of transgenic tomato plants in which two genes from the central metabolism, phosphoenolpyruvate  carboxylase (EC.3.4.1.1) and malate synthase (EC 2.3.3.9) were silenced by antisens and RNAi strategy.  1H-NMR metabolomic profiles of methanol-d4 D2O buffer extracts of tomato fruit flesh were acquired and  subjected to unsupervised multivariate statistical analysis. 1H-NMR spectra were binned into variable-size  spectral domains, making it possible to get an overall analysis of a large number of resonances, even in the  case of uncontrolled variation of the chemical shift. Principal component analysis was used to separate groups  of samples and to relate known and unknown metabolites to transgenic events. The screening of 100 samples,  from extraction to data mining, took 36 h. Thus, this procedure allows the rapid selection of metabolic  phenotypes of interest among about 30 transgenic lines.Key words: Metabolome, GMO, tomato, fruit, 1H-NMR profiling, screening

Régénération de la mandarine commune (Citrus deliciosa Ten.) par embryogénèse somatique en milieu liquide. Fusions somatiques et essais de transformation génétique

Dans le cadre du programme d'amélioration des agrumes du CIRAD-FLHOR, un procédé de culture cellulaire en milieu liquide a été développé pour une espèce modèle, la mandarine commune Citrus deliciosa Ten. Ce travail de thèse décrit les différentes étapes d'obtention d'embryons somatiques en milieu liquide : mise en place des suspensions cellulaires, contrôle et optimisation de l'embryogénèse somatique par le galactose et par "l'effet densité". Une seconde partie traite des manipulations génétiques pouvant être envisagées à partir d'un système de culture cellulaire efficace. Deux techniques complémentaires ont été abordées : la fusion de protoplastes et le transfert de gènes dans des protoplastes. Après fusion de protoplastes, des régénérations de jeunes plantes, probablement cybrides en majorité, ont pu être obtenues, tandis que l'étape de régénération a échoué dans les expérimentations de transformation génétiqu