Blanc SVSE 6 - Sciences de la vie, de la santé et des écosystèmes : Génomique, génomique fonctionnelle, bioinformatique, biologie systémique

Functions of glutaredoxins in Iron and REdox Sensing and signaling in plants – FIRES

Study of stress response in plants

Iron is an essential element for numerous physiological processes or metabolic pathways but it can be toxic under some circumstances te can generate oxidative stress. This project aims at investigating some protein families involved in cellular iron sensing and signalling.

Understand the role of glutaredoxins and BolA proteins in iron metabolism

Iron containing proteins are essential for many metabolic pathways or cellular processes (photosynthesis or respiration). However, regulation mechanisms of iron homeostasis and the functioning of iron-sulfur cluster assembly machineries in plants are not very well understood. This project aims at understanding the role of glutaredoxins and BolA proteins in these phenomenon based on genetic studies conducted in the yeast Saccharomyces cerevisiae showing the involvement of these two protein families in the regulation of iron metabolism. Understand the molecular mechanisms controling its assimilation, compartmentation and more generally its use is an important step to counteract problems linked to mysfunctioning or to variations of environmental constraints. The description of these mechanisms in the model plant, Arabidopsis thaliana could allow transfering the knowledge acquired to important agronomic species since these mechanisms are likely conserved. In particular, plants represent one of the major sources of iron for some populations that do not have access to animal sources. It illustrates the need to finely describe the cellular pathways regulating iron metabolism.

A functional genomic strategy was carried out to characterize all members (4) of each protein family, combining genetic and physiological (study of mutant or overexpressing A. thaliana lines), cellular (study of the sub-cellular localization and interaction between members of these families) and biochemical and structural (study protein-protein interactions, solve the 3D structures of the proteins alone or in complex, search for physiological partners) approaches.

The study of mutants in which gene expression is disrupted allows understanding the function of the encoded proteins. The characterization of A. thaliana lines mutated for GrxS17 showed that this glutaredoxin is implicated in cell division when plants are grown at high temperature or under a long photoperiod. Hence, defects in root, leaf and flower development have been observed.
Concerning Nfu3, the study of such mutants showed that the protein is involved in he functioning of the photosynthetic apparatus, likely in the assembly of iron sulfur containing proteins located in photosystem I. These results allow increasing the fundamental knowledge for the scientific community interested in the field.

The exact understanding of cellular and molecular mechanisms controlling iron metabolism is absolutely required to develop efficient strategies to counteract conditions of iron starvation or excess for plants. Moreover, it seems that most of these mechanisms are conserved. It is for example known that several human diseases are linked to the mysfunctioning of iron sulfur proteins. So, the results obtained in the course of this project could allow to understand the molecular mechanisms responsible of these defects and thus to find means of control against these diseases.

At mid-term of this project, the results have been valorized by 6 oral presentations in international congresses and by the publications of two research articles (Wang et al Glutathione regulates the transfer of iron-sulfur cluster from monothiol and dithiol glutaredoxins to apo ferredoxin. Protein Cell, 2012, 3(9):714-21 et Riondet et al GRXC1: a dicotyledone-specific glutaredoxin is able to stock an iron-sulfur cluster in vivo. Plant Cell Environ, 2012, 35, 360-373). Four other publications are in the process of writing.

Iron is an essential element for the nutrition of plants. Despite being one of the most abundant metals on earth, higher plants have difficulty in assimilating it because iron can generate toxic reactive oxygen species (ROS) in the reduced form (Fe2+) and is highly insoluble at neutral pH in the oxidized form (Fe3+). Iron nutrition is thus a sort of a headache for land plants and iron metabolism (uptake, transport, storage) is tightly controlled to prevent its toxicity. An imbalance in iron homeostasis results in oxidative stress in plants as well as in other organisms. In cells, iron is essential for the building of metalloproteins, essentially iron-sulfur (Fe-S) proteins and hemoproteins, which are mostly located in electron transfer chains. Fe-S proteins are required for many essential processes for life, such as photosynthesis, respiration or nitrogen and sulfur assimilation, but the various pathways of Fe-S cluster assembly and biogenesis in plants are still poorly unravelled, although some proteins homologous to those of bacterial, yeast and mammalian assembly machineries have been identified. In Saccharomyces cerevisiae, glutaredoxin 5 (Grx5), initially only considered as a small ubiquitous oxidoreductase involved in the reduction of disulfide bonds, was most likely implicated in the transfer of pre-assembled iron-sulfur clusters from scaffold to acceptors, serving as a carrier protein. The expression of orthologous Grxs from other organisms in the yeast grx5 mutant can restore the defects in Fe-S cluster assembly and the sensibility to oxidants. On the other hand, Grxs from several organisms, including plants, can bridge a [2Fe-2S] cluster into holodimers, and a plant Grx was shown to transfer it efficiently to a recipient protein. All these data suggest that Grxs could potentially function as scaffold or carrier proteins and that this role would be conserved between kingdoms. Another link between iron and Grxs came from studies in yeast demonstrating that Grxs3 and 4 form a complex with proteins of the BolA and aminopeptidase P families, which regulate the major iron responsive transcription factor called Aft1. It has been proposed that the complex responds to a mitochondrial signal and could thus regulate the cellular iron homeostasis. Our objective is thus to develop a project concerning the roles of plant Grxs (i) in iron homeostasis, either in the Fe-S cluster biogenesis machineries or in the iron sensing mechanism, and (ii) in oxidative stress conditions arising from the presence of iron and ROS. Among this multigene family in plants (around 30 genes), it is important to identify the Grx(s) which is (are) involved in these processes and their subcellular localization, as iron and ROS are present in most sub-cellular compartments and as different assembly systems are present in mitochondria, chloroplasts and cytosol. For this purpose, we will develop a multidisciplinary approach (genetics, physiology, cellular biology, biochemistry) using Arabidopsis thaliana as a model plant. The function of a specific class of Grxs able to incorporate Fe-S clusters will be assessed using transgenic plants (extinction or overexpressing lines) grown under different iron supply (deprivation or excess) or under different light/temperature regimes. The transcriptome analysis of the plants displaying a macroscopic phenotype should help identify pathways and target genes/proteins potentially regulated by Grxs. In addition, the Grx-interacting proteins identified in the course of this project by yeast two hybrids, co-immunoprecipitation or affinity studies as well as candidate proteins of the BolA family, will be produced as recombinant proteins for their biochemical analysis and functional characterization.

Project coordinator

Monsieur Nicolas ROUHIER (UNIVERSITE DE NANCY I [HENRY POINCARE]) – nrouhier@scbiol.uhp-nancy.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CEA COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - DIRECTION DU CENTRE DE FONTENAY-AUX-ROSES
IRD / CNRS/ UPVD INSTITUT DE RECHERCHE POUR LE DEVELOPPEMENT - IRD
UMR Iam UNIVERSITE DE NANCY I [HENRY POINCARE]
BPMP INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE - CENTRE DE MONTPELLIER

Help of the ANR 500,000 euros
Beginning and duration of the scientific project: - 36 Months

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