Legume-Rhizobium Symbiosis: cellular redox status

Background, Research topics and objectives

The growth of cultivated plants largely depends on the presence of a sufficient nitrogen quantity in soil. The massive use of nitrogen fertilizers in intensive farming is a considerable risk for the environment, leading to major pollution of the environment by nitrate and nitrite. Among plant-microbe interactions, the symbiosis between nitrogen-fixing rhizobia and legumes represent a major opportunity for the rational management of nitrogen inputs in agriculture and for the reduction of the soil pollution. During symbiosis, essential cellular functions of plant cell are manipulated by the symbiotic bacteria in order to allow its penetration. This leads to the formation of a new organ, the so-called nodule, capable of fixing atmospheric nitrogen and thus allowing the plant to meet most of its nitrogen requirements. However, this symbiosis is very sensitive to environmental changes and its lifespan is limited: the developmental nodule senescence, a breakdown of the symbiotic association, appears before the plant senescence. Thus, one major characteristic of the relationship between both partners is a switching from a compatible to an incompatible interaction.The detailed understanding of mechanisms involved in the establishment and the maintaining of this benefic symbiotic relation is essential for the development of strategies to improve plant health and contribute to more environmentally-friendly agricultural practices.

Using the Medicago truncatula/Sinorhizobium meliloti interaction as symbiotic model, the research conducted in our team aims at studying the role of the cellular redox state in this interaction and deciphering mechanisms leading to the nodule senescence in both partners. Within this context, the roles of glutathion (GSH), hydrogen peroxide (H₂O₂) and nitrogen monoxide (NO), three major molecules involved in the regulation of the cellular redox state, are analyzed. In parallel, the importance of plant cysteine protease and bacterial toxin-antitoxin systems is investigated during nodule senescence

Fig. 1 : Study of symbiotic Rhizobium-legume interaction

A. nodule meristem Medicago truncatula.
B. fixing nodule mature.
C. senescent nodule (zone IV called senescence zone).
D. Study of the expression of catalases of S. meliloti by the promoter-reporter gene fusions in the functional nodule.

Biological models studied

Legumes: Medicago sativa, Medicago truncatula and others Legumes
Symbiotic bacteria: Sinorhizobium meliloti 

Scientific expertise

The team has long-standing experience of the nitrogen fixation process and of the involvement of reactive oxygen species in it. However, very few data exist and few studies have been devoted to the importance of the cellular redox state in symbiotic legume/bacteria interactions. Moreover, nodule senescence (aging) is a mechanism that has received little attention and is therefore not yet well understood.

Current priorities

What is the role of the molecules involved in the regulation of the cellular redox state (H2O2, NO, GSH) in nodule establishment, function and senescence?
More specifically:
What is the role of H₂O₂ in the establishment and function of symbiosis (molecular target, origin and dynamics of H₂O₂ production, etc.)?
What are the mechanisms regulated by GSH and NO in the formation and maintenance of nodules?
What is the contribution of the bacteroid to nodule senescence?

Fig. 2 : Study of symbiotic Rhizobium-legume interaction
E. Visualization of Bacteroids by electron microscopy during salt stress.
F. Study of the expression of ferritin in a salt stress by in situ hybridization.
G. Characterization of gene expression and synthesis of glutathione homoglutathion by northern blot.

Application

To reduce the quantity of nitrogen fertiliser used on crops by improving the natural fixation of atmospheric nitrogen produced through symbiosis. 

Scientific partnerships and financing

Bekki A.Laboratoire de Biotechnologie des Rhizobiums et Amélioration des Plantes, Département de Biotechnologie, Université d’Oran Es Senia, Oran, Algeria.

Rouhier N. et Jacquot J.-P.UMR INRA UHP 1136, Interactions Arbres Microorganismes, IFR 110 EFABA, Université de Lorraine, Faculté des Sciences, BP 70239, 54506 Vandoeuvre cedex, France.

Mergaert P.Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France

Frugier F. and Gruber V.Institut des Sciences du Végétal (ISV), Centre National de la Recherche Scientifique, Gif-sur-Yvette, France

Bruand C. et Meilhoc E.Laboratoire des Interactions Plantes-Microorganismes (LIPM), INRA, UMR441, 24 Chemin de Borde Rouge–Auzeville, CS 52627, F-31326, Castanet-Tolosan Cedex, France

Bonfante P. et Lanfronco L.Université de Turin. Dipartimento di Scienze della Vita e Biologia dei Sistemi Turin, Italie.

Wifp D, Jeandroz Sylvain.UMR INRA/UB/Agrosup Dijon Agroécologie Dijon.

Delledonne M.Dipartimento di Biotecnologie, Universita' degliStudi di Verona, Italie.

Dusha I.Institute of Genetics. Laboratory of Medicago Genetics. Biological Research Centre. Hungarian Academy of Sciences.6726 Szeged, Temesvárikrt. 62.

Smiti S., Unité de Recherche en Ecologie végétale, Université El Manar de Tunis, Tunis, Tunisie.

Gojon A., Sentenac H., UMR Biochimie et Physiologie Moléculaire des Plantes CNRS/INRA/SupAgro /UM2, Montpellier, France.

Rolin D., UMR Biologie du Fruit et Pathologie, INRA, Université de Bordeaux I et Bordeaux II, Villenave d'Ornon, France.

Ghoulam C,Université de Marrakech. Equipe de Biotechnologie Végétale et Agro-physiologie des Symbioses, Marrakech, Maroc.

Major results

Development of new cellular tools to analyze the symbiotic nitrogen fixing interaction in vivo.

In order to better understand the  nitrogen fixing symbiosis, we have developed new cellular tools. In this context, multiple probes analyzed using confocal microscope imaging were developed and used in nitrogen fixing nodules. PBS pH was measured in vitro during the whole symbiotic process using ratiometric fluorescent probe. This analysis showed that nitrogen fixing zone maturation goes with the acidification of the peribacteroid  space in the nitrogen-fixing organite, the symbiosome (Pierre et al., 2013). The viability of bacteroids was analyzed during their differentiation and throughout the symbiotic interaction in vivousing nodule sections with the Live/Dead^® BacLight^TM probe. Finally, the in vivo quantification of H₂O₂ was measured in nodule using a protein probe HyPer. This analysis correlates the production of H₂O₂ with the regulation of MtSpk1, an H₂O₂ regulated gene (Andrio et al., 2013).      

 

Identification and characterization of H₂O₂ and NO-regulated genes in the rhizobial symbiosis.

Reactive oxygen species (ROS), particularly H₂O₂, and nitric oxide (NO) play an important role in signalling in various cellular processes. The involvement of these molecules in the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction raises questions about their effect on gene expression. Transcriptomic analyses were performed on inoculated roots of M. truncatula to identify genes regulated by these two molecules. Numerous genes were found to be differential regulated in H₂O₂ and NO-depleted roots during the symbiotic interaction.  Characterisation of candidate genes such as MtSpk1, a putative protein kinase, showed their involvement in the signal transduction pathway involved in the symbiotic interaction  (Andrio et al., 2013 ; Boscari et al. 2013).