Abstract
Manganese (Mn) is an essential metallic micronutrient for all living organisms. In plants, it acts as a cofactor for many enzymes involved in major metabolic pathways. However, the bioavailability of Mn in the soil is variable and its deficiency or excess can be limiting for plant growth. In order to adapt to these fluctuations, plants benefit from many transporters controlling root absorption of Mn and its distribution to the various organelles of the cell. The maintenance of Mn cellular homeostasis relies notably on the regulation of the localization or activity of these transporters. In Arabidopsis thaliana, high-affinity Mn uptake is mainly mediated by the transporter NRAMP1, whose subcellular localization depends on the metal availability. NRAMP1 is stabilized at the cell surface under Mn starvation and rapidly cycles between the plasma membrane and the endosomes in response to Mn excess through a phosphorylation-dependent mechanism. Based on this knowledge, my thesis work aimed to deepen the characterization of NRAMP1 post-translational regulatory mechanisms in response to Mn in Arabidopsis. The objective of my first research axis was to determine the involvement of ubiquitination in the regulation of NRAMP1 and to identify the enzymes and transporter residues involved in this process. My second research axis aimed to identify kinases controlling NRAMP1 phosphorylation and to characterize the context and the role of these mechanisms in the regulation of the transporter in response to Mn. During my thesis, I used a wide spectrum of biochemical, proteomic and site-directed mutagenesis strategies to answer these questions. In order to identify enzymes involved in the ubiquitination or phosphorylation of NRAMP1, I sought for protein partners of the transporter through interactome-based untargeted approaches and candidate gene approaches. The latest focused on the calcium-activated CIPKs and CPKs kinases families and established CIPK23 as a main candidate. In vitro phosphorylation experiments coupled with reverse genetics approaches allowed me to demonstrate that CIPK23 phosphorylates Ser20 and 499 on the N- and C-termini of NRAMP1. Physiological analyzes led me to the conclusion that CIPK23 is likely to control the activity of the transporter depending on the Mn status of the plant. Thus, my research on NRAMP1 regulation by phosphorylation and ubiquitination has contributed to a better understanding of the complex mechanisms supporting Mn homeostasis in plants.