regulation of DenItrificatiOn by Root Exudates – DIORE

The nitrogen fertilizer used for crop production is partially emitted as N2O, a gas having about 300 times more impact on global
warming than CO2. N2O is generated during microbial nitrification and denitrification, which are common in agrosystems. The whole
denitrification process requires optimal conditions in terms of oxygen pressure, availability of readily assimilable carbon source, and
the presence of nitrate. Although, the importance of carbon source in this process is well known, very little is known with respect to the
microbial pathways through which N2O reduction is favoured.
In bacteria, the “Carbon Catabolite Repression” (CCR) mechanism is a complex regulatory process allowing bacteria in the presence of
different carbon sources, to assimilate the preferred one and to inhibit the expression of genes encoding enzymes involved in the use of
the non preferred carbon sources and other genes involved in different functions. This mechanism enables colonization of very different
niches and adaptation to changing environments. So far, two types of CCR mechanisms were described: CCR mechanism related to the
preference for glucose, as the case for B. subtilis, and reverse-CCR (rev-CCR) related to the preference for organic acids, such as in
Pseudomonas, which abolishes the paradigm that glucose is “the easy & preferred molecule”.
The present project aims at investigating the role of catabolic repression in the course of all the steps of the denitrification process under
natural conditions. For this, we will start by using the model strain Pseudomonas brassicacearum, exhibiting rev-CCR mechanism, to
validate the link between CCR and denitrification function and to characterise the regulators including proteins and non-coding small
regulatory RNAs that may be involved in the regulation of denitrifying genes, by genetic and transcriptomic approaches. A second
objective is to link the denitrification activity to the nature of carbon source in the rhizosphere. Therefore, we will use Arabidopsis
thaliana mutants altered in root exudation of different primary metabolites, to evaluate the influence of the quality and the quantity of
primary metabolites on the regulation of the expression of the denitrification key players, and to determine if in vitro observations occur
under natural conditions and if they can be generalized to bacterial communities in the rhizosphere. The last objective is to apply root
exudates primary metabolites as marker to select canola lines cropping plants that reduce microbial N2O emission and to analyse the
microbiota of the most contrasting lines. An interdisciplinary partnership has been assembled with expertise in bacterial ecology
(physiology & genetics), plant-microbe interactions, nitrogen cycle and plant metabolomics to reach these goals.
The discovery of how denitrification is regulated under natural conditions has an tremendous ecological interest and could lead in the
future to various environmental applications such as inhibiting N2O-producing (particularly denitrification) and stimulating N2Oreducing
bacteria in the environment by plant primary metabolites to minimize N2O emissions and N Loss. Indeed, the global N2O
emissions from cultivated soils have been estimated at 3.3 Tg N2O yr-1 (Lars et al., 2012) and few tools are available to reduce the
emissions from natural ecosystems, apart from reducing the inputs of anthropogenic reactive nitrogen. This project will also allow us to
predict N2O emissions and propose new criteria of selection of agricultural crops based on the nature of exuded primary metabolites
that minimize N loss and N2O emission to the atmosphere.