Etude des Régulations Post-traductionnelles du Transporteur Racinaire de NO3- NRT2.1 – TransN

Nitrogen is the mineral nutrient required in the highest amounts by plants and is most frequently limiting for growth and yield, leading to massive use of N fertilizer in agriculture. In higher plants, the molecular basis of root NO3- uptake has been the matter of intensive studies during the last decade. One main outcome of all these studies is the identification of NRT2.1 as a main component of the root high affinity NO3- uptake system (HATS) in Arabidopsis. Furthermore, it has been shown that NRT2.1 not only plays a role in NO3- transport, but also acts as a sensor or transducer governing lateral root growth responses to NO3-. The regulation of NRT2.1 has been thoroughly studied showing a strong correlation between NRT2.1 expression and HATS activity. However, despite its central role in plant nutrition, the knowledge concerning the regulatory mechanisms involved is mostly restricted to the mRNA level, as it is the case for most root ion transporters to date. Because of the technical problems associated with the study of intrinsic membrane proteins, much less information is available concerning the regulation of NRT2.1 at the protein level. Until now, only one report from the coordinator of this project has addressed this question. This study indicated that in addition to transcriptional regulation, posttranslational regulation of NRT2.1 plays an important role in modulating the activity of this NO3- transporter. It revealed that (i) NRT2.1 is part of high molecular weight (MW) complex (~120 kDa), which co-exists with the monomeric form (~45 kDa) in the plasma membrane, (ii) The abundance of NRT2.1 protein in the plasma membrane is only slowly affected by environmental factors know to affect both NRT2.1 mRNA and NO3- HATS activity and (iii) there is evidence for partial proteolysis of NRT2.1 at the C-terminus of the protein. The project presented here aims at taking advantage of the new developments in biochemical and proteomic tools and approaches for elucidating the mechanisms involved in the posttranslational control of NRT2.1. We propose to take a systematic approach with the aims to: (i) identify proteins interacting with NRT2.1, (ii) determine the site and the role of NRT2.1 C-terminal cleavage and (iii) determine if NRT2.1 is phosphorylated in response to environmental signals like photosynthesis and N starvation. This will allow us to unravel the precise nature of the various NRT2.1 forms and to manipulate them independently for investigating their specific function in NO3- uptake and root development. In addition, the identification of posttranslational events affecting NRT2.1 in the plasma membrane (cleavage in C-terminus, phosphorylation) will provide insights on the regulatory mechanisms responsible for the fast modulation of the activity of this major transport system in response to environmental changes. This work will be done in the context of a collaboration between the group of Dr. Alain Gojon (partner 1) in the Department of Biochimie et Physiologie Moleculaire des Plantes in Montpellier and the group of Dr. Michel Rossignol (partner 2) in the Laboratoire de Proteomique Fonctionnelle in Montpellier. These two groups have a leading activity in the field of NO3- transport in plants and proteomic respectively, and the goal is to combine their complementary expertise. Partner 2 will be involved in (i) the extensive use of GC/MS MS, LC/MS MS and MALDI TOF to investigate post-translational modifications, such as phosphorylations, using the protocols they developed for their proteomic platform and (ii) the development of methodologies like Blue native PAGE to study protein-protein interactions, or C-terminus sequencing strategies to determine NRT2.1 cleavage site. The outcome of theses approaches will be used by Partner 1 for functional characterisation in a set of environmental signals known to affect the activity of NRT2.1 transport system. This will involve the development of transient transformation of Arabidopsis thaliana roots and the use of Xenopus Oocytes. By joining our efforts we are expecting to decipher new mechanisms controlling the main entrance gate for nitrogen into plants.