ChAnnels and Reactive Oxygen species in Legume root hair: role in Symbiosis with Rhizobium – CAROLS

The root hair plays crucial roles in water and nutrient uptake, and thus in plant development. In Legumes, it also plays a central role in the establishment of the nitrogen-fixing symbiotic interaction with rhizobia, which has a crucial importance for sustainable agriculture as it largely contributes to limitation of nitrogen fertilizers. Furthermore, the root hair provides a model to investigate the biology of a single differentiated plant cell type, one of the present challenges for plant scientists, and is considered as a highly promising model for plant cell systems biology.

A hallmark feature of the root hair, shared with very few cell types (pollen tube, fungal hyphae and animal axons), is its polarised cell growth, occurring only at the tip. This so-called tip growth is a highly complex process, in which ion channels and reactive oxygen species (ROS) are key actors. Furthermore, these actors play interacting roles: channel activity can be controlled by ROS involving signals and, reciprocally, signalling ROS production can depend on channel driven ion fluxes. Evidence is available that this ion channel-ROS mediated control of root hair tip growth, initially observed in the model plant Arabidopsis thaliana, is also active in Legumes. Moreover, in the latter case, re-orientation of the tip growth occurs during the initial physical interaction with the rhizobial microsymbiont, an early step in the establishment of the nitrogen-fixing symbiosis: bacterial infection is initiated by attachment of the bacteria to the root hairs that curl, due to this re-orientation.

The aim of the CAROLS project is to obtain a holistic view of the involvement of root hair plasma membrane ion channels and of their regulation by ROS during the first steps of the symbiotic interaction between the model Legume Medicago truncatula and its microsymbiont Sinorhizobium meliloti. The work will be divided into six tasks: (i) in situ electrophysiological analyses, providing the functional repertoire of the ion conductances active in the plasma membrane of M. truncatula root hairs, (ii) molecular analyses and cloning, providing the molecular repertoire of the ion channel genes expressed in root hairs, (iii) verification that the channels selected during task (ii) are actually present in the root hair plasma membrane, (iv) functional characterisation of cloned channels in heterologous systems, (v) analyses of the role of ROS in the ion channel regulation and (vi) in planta analyses of selected mutant plants (affected in channel activities or ROS production) and phenotyping of their root hair growth and symbiotic abilities. By combining up-to-date electrophysiology techniques (laser dissection coupled to patch-clamping, a methodology that is already available in the consortium) with molecular, genetic and functional analyses, the project will provide the first systematic and integrated study of the root hair plasma membrane ion channels. In particular, through the use of the laser dissection technique, it will allow to study the ROS effects on channel activities at various stages of root hair growth, including root hair curling and infection thread initiation. These innovative methodology and device, which may be unique at the international level, give to the project an ideal situation in the framework of the international competition. Moreover, the project will benefit from the almost completed sequencing of the M. truncatula genome. It will lead to important breakthroughs in the analysis of the roles of ion channels and their regulation by ROS in root hair tip growth and interaction with symbiotic bacteria. Finally, at a still wider level of biological concerns, it will open new research lines in the analysis of the interplay between ROS and ion channels in plant signalling and adaptation to biotic and abiotic environmental conditions, root hair physiology and systems biology.