"Nitrate-NO" respiration study in symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti : searching for mitochondrial nitrite transporter

Marianne Prevot

Abstract:

Symbiosis between legume plants and nitrogen-fixing bacteria (rhizobium) constitute a major issue to reduce nitrogen fertilization in crops. Central function of nodules is the enzymatic reduction of atmospheric nitrogen (N2) into ammoniac (NH4+) by the bacterial nitrogenise, and the subsequent assimilation in the plant cell. This highly energy-consuming process is a paradox as occurring in hypoxied tissue where mitochondrial respiration -the main ATP source- is strongly reduced. The question rose how the ATP required for cellular functioning is generated into nodules. Based on recent observations on hypoxied roots, we rose the hypothesis that, in nodules, ATP is formed trough a cyclic respiration -called nitrate-NO respiration- that consists in four steps: 1) nitrate reductase (NR) reduces nitrate (NO3-) into nitrite (NO2-) in the cytosol ; 2) NO2- is transported from cytosol into mitochondria using transporter(s) protein(s) ; 3) electrons of mitochondrial transfer chain reduces NO2- into nitric oxide (NO), thus allowing respiration functioning and ATP formation ; and 4) NO freely diffuses from mitochondria to cytosol to be oxidized back into NO3- through leghemoglobine (Lb) action. During this thesis, we showed, using Medicago truncatula-Sinorhizobium meliloti system as a model, that NRs and electron transfer chains from both plant and bacterial partners were involved in NO production of functioning nodules. NR activity was also showed to be crucial to maintain high energetic status required for nitrogen fixation. The second aim was to characterized nitrite transport across purified root mitochondria. Then, mitochondrial nitrite transporter candidate(s) were investigated. An in silico strategy searching for predicted proteins in M. Truncatula genome, potentially localized to mitochondria, that shared homologous sequences with known transporters was conducted. 6 candidates were chosen at the end of this analysis. Membrane mitochondria proteins were identified using mass spectrometry (MS) analysis, revealing 3 new candidates. The expression profile in normoxied and hypoxied roots and nodules was characterized for the 9 candidates retained. Two of them showed particularly promising patterns. Finally, sub-cellular localization and tissular expression during nodule development were analyzed for some candidates that shared interesting expression patterns.

Journal:

Thesis of the University of Nice-Sophia Antipolis

Link:

https://www.theses.fr/2012NICE4099