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Accueil > Départements > Biologie Cellulaire > Sébastien THOMINE : Approches Intégratives du Transport des Ions

NEW : STAGES M2

Nous proposons pour 2018 les sujets de stage M2 suivants à l’Ecole Doctorale Sciences du Végétal, Université Paris-Sud :

- Deciphering the molecular and cellular mechanisms of iron storage and remobilization in seeds (Sebastien Thomine) click to read

- Towards the identification of metal uptake transporters involved in nickel hyper accumulation in plants (Sylvain Merlot) click to read

- Life and death of metal transporters : role of the evolutionary conserved protein AtPH1/2 (Michele W. Bianchi) click to read

- Rôle des canaux mécanosensibles dans la transduction mécanique chez Arabidopsis thaliana (Jean-Marie Frachisse) click to read

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You are welcome to contact the corresponding investigator for additional information.

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Deciphering the molecular and cellular mechanisms of iron storage and remobilization in seeds.

In plants, iron (Fe) fulfills many functions as the cofactor of a wide range of proteins involved in photosynthetic and respiratory electron transfer chains and biosynthetic pathways. Iron acquisition by plants is not only crucial from an agronomical point of view, but also for human health since seeds represent the main dietary source of iron for humans. More than 2 billion people are affected by Fe deficiency, caused by the consumption of grains with low quantity and availability of Fe. Biofortification (ie increasing vitamins and micronutrient contents by breeding or biotechnologies) has been proposed as a sustainable solution to this problem (Murgia et al., 2012, TIPS 17(1), 47-55). The major bottleneck of biofortification strategies is the lack of knowledge on the genes involved in the control of Fe storage and availability in seeds. The objective of the project is the identification and the characterization of new genes controlling iron storage and remobilization in seeds using a genetic approach in the model plant Arabidopsis thaliana.

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Towards the identification of metal uptake transporters involved in nickel hyper accumulation in plants.
Transition metals such as Iron, Zinc (Zn) or Nickel (Ni) are essential for plants but generate oxidative and genotoxic stresses at high concentration. However, more than 500 plant species, growing on metalliferous soil, are able to accumulate tremendous amount of metals in their shoot, including the model hyperaccumulator Noccaea caerulescens related to Arabidopsis thaliana. There is a regain of interest to study metal hyperaccumulators because they are instrumental to develop sustainable phytotechnologies such as phytoremediation and phytoextraction to extract metals from soil and valorize their metal enriched biomass (Rascio & Navari-Izzo 2011). To support the development these technologies it is important to better understand the molecular mechanisms involved in metal hyperaccumulation in plants. We have previously targeted our studies on the molecular mechanisms involved in the storage of metals in leaves (Merlot et al. 2014 ; Sanchez Garcia de la Torre, unpublished) and now would like to investigate the mechanisms taking place in roots that are key for metal uptake and translocation to the shoot. We have previously conducted an extensive RNA-seq study of several N. caerulescens accessions with contrasting hyperaccumulation capacity and specificity for Ni, Zn and Cd, to identify genes linked to these traits. Our primary analysis of the RNA-Seq data revealed that the orthologs of the root metal transporters IRT1 and NRAMP1 are candidate genes implicated in Ni hyperaccumulation. The function of IRT1 and NRAMP1 in the regulation of iron homeostasis in Arabidopsis thaliana is very well documented (Castaings et al. 2016 ; Agorio et al. 2017), but their potential role in the efficient uptake of Ni in the metal hyperaccumulator N. caerulescens is very original. The aim of this project is to validate the role of the IRT1 and NRAMP1 orthologs in Ni hyperaccumulation using a combination of bioinformatics analysis and functional studies in yeast and transgenic N. caerulescens plants.

The first step of the project will be to perform an in-depth bioinformatics analysis of the available RNA_seq data to analyze the expression level and presence of polymorphism in the coding sequence of the IRT1 and NRAMP1 orthologs in accessions of Noccaea caerulescens with contrasting metal accumulation capacity. The goal of this analysis will be to establish a correlation between the expression level (and/or the presence of polymorphism) of IRT1 and NRAMP1 orthologs and the Ni hyperaccumulation capacity of the corresponding accessions.
IRT1 and NRAMP1 orthologs will be cloned from a Ni hyperaccumulator accession (e.g. N. caerulescens firmiensis) and overexpressed, with and without a fluorescent protein tag, in roots of an accession that do not accumulate Ni (e.g. N. caerulescens La Calamine). In parallel, IRT1 and NRAMP1 orthologs will be silenced in roots of a Ni hyperaccumulator accession using artificial microRNA technology (amiRNA). The root transformation of N. caerulescens by R. rhizogenes is mastered in our team and it takes about 8 weeks to regenerate transgenic roots for further analyses. The expression level IRT1 and NRAMP1 orthologs in transgenic roots will be analyzed by qRT-PCR and the impact of the modification of IRT1 and NRAMP1 expression (over expression and silencing) on Ni accumulation in roots and leaves will be measured by atomic emission spectrometry (MP-AES). The localization of IRT1 and NRAMP1 orthologs fused to a fluorescent protein will be analyzed in transgenic roots of N. caerulescens by confocal microscopy. During the regeneration of transgenic roots, IRT1 and NRAMP1 orthologs will be expressed in yeast to study their capacity transport Ni and other metals using complementation, growth sensitivity assay and direct measurement of metal accumulation using MP-AES. These assays are routinely performed in our laboratory (Merlot et al. 2014).
If this is required because of time constrains, we will focus on the functional analyses of the ortholog of one metal transporter. During this Master training, the student will acquire experience in several techniques that will be essential to develop her/his future project in molecular genetics. This Master project can be further developed in the frame of a PhD thesis.

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Life and death of metal transporters : role of the evolutionary conserved protein AtPH1/2.
To achieve metal homeostasis, plants regulate the activity of metal transporters and their cellular localization and trafficking across cell compartments.

Recently, we have discovered that mutations in AtPH1 perturb the localization of the manganese and iron transporter AtNRAMP1 of Arabidopsis, inducing its accumulation on the vacuolar membrane (PNAS 2017, E3354-E3363). AtPH1 is therefore a new regulator of the cellular trafficking of metal transporters. In parallel, we have also determined that the abundance at the plasma membrane of AtNRAMP1 is controlled by manganese availability (ongoing work).

AtPH1 specifically binds phosphatydil inositol 3 phosphate (PI3P). This lipid is abundant in the membranes delimiting specific cell compartments of the endocytic and vacuolar degradation pathways, where it is thought to serve as anchor to specific proteins. The precise function of PI3P in these compartments is under intense investigation. The Arabidopsis genome also contains a paralogue of AtPH1, which we named AtPH2.

Interestingly, no information is available in other organisms on the molecular or cellular role of the homologs of AtPH1/2, which therefore can be seen as a paradigm to elucidate the role of these PI3P-binding, membrane-associated proteins in eukaryotes.

The aim of this project is to investigate the cellular mechanism by which AtPH1 controls the fate of the AtNRAMP1 transporter, and the physiological importance of AtPH1 at the plant level. The student will exploit available mutants and fluorescent reporter lines, and contribute to the generation of new tools.

Cellular role of AtPH1/2.
AtPH1/2 may affect the protein stability of AtNRAMP1 and/or its trafficking towards the vacuole. To test the role of AtPH1/2 in AtNRAMP1 stability, the student will analyze protein and mRNA levels of AtNRAMP1-GFP fusions driven by the constitutive ubiquitin promoter in WT and atph1atph2 plants. These observations will be done separately for roots and leaves, in the presence and absence of Mn. The intensity and cellular pattern of AtNRAMP1-GFP fluorescence will be monitored in the same conditions, by confocal microscopy.
To test the role of AtPH1/2 in AtNRAMP1 trafficking from the plasma membrane to the vacuole, the co-localization of AtNRAMP1-GFP with AtPH1-mCherry, and with markers of pre-vacuolar compartments, will be evaluated in WT plants, in conditions likely to stimulate (excess metals) or inhibit (Mn starvation, pharmacological inhibitors) this trafficking pathway.

AtPH1/2 implication in metal homeostasis at the plant level.
atph1atph2, atph1, atph2 and wt plants will be grown in vitro in different conditions of Mn and Fe availability. Phenotypes will be compared, focusing on mass of aerial parts, photosynthetic activity, survival rate and length/architecture of the root system.

Local context : MWB (Plant Cell 2010, 1575-91 ; Nature Comm 2014,5:4121), collaborates closely on this topic with Sylvain Merlot and Sebastien Thomine within the « Integrative Approaches to Ion Transport » group at I2BC.

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Rôle des canaux mécanosensibles dans la transduction mécanique chez Arabidopsis thaliana

Les plantes terrestres présentent une réponse au toucher. Parmi les plus spectaculaires on peut citer celle de Mimosa pudica et de la Dionée gobe-mouche qui se referment quand on les touche. Les mécanismes moléculaires de cette réponse restent peu connus. Des acteurs potentiels dans la voie de transduction mécanique sont les canaux mécanosensibles (Hamilton et al., 2015 ; Peyronnet et al., 2014). Ces protéines transmembranaires sont des canaux ioniques qui sont activés par une augmentation de la tension mécanique de la membrane. Elles se comportent donc comme de véritables petits capteurs capables de coupler une variation de force à une variation de flux d’ions au travers de la membrane.
Peu de canaux mécanosensibles ont été identifiés et caractérisés à l’heure actuelle chez les plantes et leur fonctions restent à préciser. Cependant, en collaboration avec G. Ingram et O. Hamant (ENS, Lyon) nous avons récemment caractérisé au laboratoire par une étude d’électrophysiologie (Tran et al., 2017) un canal calcique mécaniquement activable (RMA : Rapid Mechanically Activated) dans la membrane plasmique de protoplastes d’Arabidopsis thaliana. La mécanosensibilité de ce canal est associé à la présence de la protéine membranaire DEK (DEFECTIVE KERNEL) qui est par ailleurs essentielle pour la coordination des étapes du développement post-embryonnaire et pour l’adhésion cellulaire (Tran et al., 2017).

Les objectifs du M2 seront (i) à l’échelle moléculaire, de caractériser le fonctionnement du canal RMA et notamment le rôle du cytosquelette dans la mécanosensibilité, (ii) à l’échelle de l’organe, de mettre en évidence et caractériser le couplage entre la stimulation mécanique et le signal calcique ainsi déclenché.

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