Signaling pathways involved in the phosphorylation of membrane proteins in response to N and C – SIPHON

Nitrogen (N) is an essential macronutrient for plant growth. In addition, N also has signaling functions by triggering specific adaptations in central metabolism and root development. While basic uptake mechanisms for N through plasma membrane located transport systems are well known and global transcriptional and post-transcriptional responses to changes in N and carbon (C) availability have been studied in the past, the detailed mechanisms in regulation of N uptake, as well as signal transduction events are still barely revealed. Our work will aim at closing this gap by particularly focusing on studying the post-translational regulation of the nitrate (NO3-) transporter NRT2.1.
Despite N- and C-dependent regulatory mechanisms for NRT2.1 have been thoroughly described, the knowledge concerning the molecular mechanisms is still poor, and most regulatory effects were studied at the mRNA level. Because of technical problems associated with studying intrinsic membrane proteins, much less information is available concerning the protein-level regulation of NRT2.1. However, within the last couple of years, the general picture has emerged that both NO3- and ammonium (NH4+) transporters are controlled at the protein level by posttranslational modification to ensure fast responses to highly fluctuating environmental conditions directly at the plasma membrane.
Phosphorylation is the most well studied post-translational modification with regulatory function. It can induce changes in protein activity, provide docking sites for protein-protein interaction or induce changes in subcellular location. Thus, modification-dependent changes in protein abundance, activity and subsequent modification-dependent protein-protein interactions are important features in signaling networks. NO3- transporters were in the past shown to be subject to protein phosphorylation, and especially for NRT1.1 the role of protein phosphorylation in switching the transport affinity has been well described. Also for NH4+ transporters, a phosphorylation-dependent activation/inactivation mechanism was elucidated in recent years. Therefore, a particular focus of this project is to study the role of protein phosphorylation for the regulation of the major root nitrate transporter NRT2.1 and the protein kinases and phosphatases involved in N and C signaling.
This work will be done in the context of a collaboration between Dr. Laurence Lejay in the group of Dr. Alain Gojon in the Department of Biochimie et Physiologie Moléculaire des Plantes (BPMP) in Montpellier (France) and the group of Dr. Waltraud Schulze, Department of Plant Systems Biology, University of Hohenheim (Germany). The scientific aim of this collaborative project is (i) to characterize the condition-dependent phosphorylation of target proteins related to N uptake and metabolism in response to C and N availability. It will involve both the identification of novel C/N regulated proteins as well as the specific characterization of the role of the known NRT2.1 phosphorylation sites in the regulation of root NO3- uptake and root development, (ii) finding candidate protein kinases and phosphatases using both NRT2.1 and selected N-metabolism related proteins as targets and (iii) characterizing in planta the impact of the interesting protein kinases and phosphatases on the phosphorylation of both NRT2.1 and selected N metabolism related proteins and on the regulation of root NO3-uptake and root development. We will also start to (iv) investigate the proteins interacting with the selected protein kinases and phosphatases as baits. We are convinced that with the complementary expertise of both partner groups and with a clear focus on NRT2.1 regulation at the protein level, we will gain novel valuable insights into a crucial plant function, namely the regulation of root N uptake.