INTEGRREGULFESPLAST

Regulation of iron-sulfur (Fe-S) cluster assembly in plastids and coordination with plant physiology

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 Obiettivo del progetto (Objective)

'Pivotal to the essential plant process of photosynthesis, which determines crop productivity, are iron-sulfur (Fe-S) cofactors. My outgoing phase host, Dr. Pilon at Colorado State University (USA), played a major role in defining the core components of the chloroplast Fe-S cluster assembly machinery essential for photosynthesis. Using an integrated biochemical-physiological-genetic approach in the model plant Arabidopsis thaliana I aim to investigate how the machinery components interact to produce Fe-S clusters, and how assembly is regulated. Specifically, I will study protein-protein interactions that mediate Fe-S assembly and use inducible RNA interference to create conditional loss-of-function mutants for individual components. This will also train me in methods developed in Dr. Pilon’s group: affinity purification, protein-protein interaction, in vitro reconstitution and RNAi. I will use these tools and skills in the return phase in the group of Dr. Gaymard in Montpellier (France) where I will establish a model for regulation of Fe-S assembly. Using the state of the art high-throughput “Phenopsis” platform I will study Fe-S assembly in the various mutant plant lines and investigate its response to changing environmental conditions, and stresses that limit agricultural productivity. This innovative project will provide basic knowledge and have additional impact for the breeding of more productive, nutritious plants. The importance of iron for food production and quality, key areas in the EU, cannot be overstated. This fellowship will contribute to European excellence and mobility via training, knowledge transfer and implementation of integrated methods. The project provides mentoring and management training; further complementary skills training is ensured by the highly interactive environments of both host labs at premier research institutes. The proposed project will provide excellent professional preparation for a group leader in the post-genomic era.'

Descrizione progetto (Article)

In the context of global climate change and dynamic environmental conditions, understanding regulation and function of Fe-S clusters in the chloroplast is critical. With this in mind, researchers initiated the EU-funded project INTEGRREGULFESPLAST.

Previous research has shown that the cysteine desulphurase enzyme, CpNifS is required for S supply during Fe-S cluster assembly in chloroplasts. The protein CpSufE binds to CpNifS to assemble Fe-S clusters. However, knowledge about how exactly chloroplasts assemble these clusters is still limited. To resolve this, researchers applied an integrated biochemical-physiological-genetic approach in the model plant Arabidopsis thaliana to study the chloroplast Fe-S assembly system.

Plants were grown in hydroponic conditions to assess their response to variations in nutrition and environment. In particular, differing Fe and S levels as well as varied lighting conditions were tested. Researchers assessed changes in physiological and phenotypic parameters such as plant biomass, chlorophyll content, photosynthetic activity and gene expression.

Using techniques such as Western-blotting and antibody collection, the scientists observed changes in target protein levels and Fe-S regulation. Inducible artificial RNA interference constructs were designed and selected KO plant mutants were used to conditionally suppress proteins and study their functions.

Project members already isolated several components belonging to certain protein families associated with Fe-S biogenesis in the chloroplast using sequence analogy. An important discovery was the interaction of the protein adenosine 5?-phosphosulphate reductase (APR) with SufE and its role in S metabolism during Fe-S cluster formation. Results revealed a depleted supply of APR proteins when Fe supply is low suggesting that cysteine desulphurase activity is Fe-dependent. However, NifS and SufE levels were unaffected by Fe levels.

INTEGRREGULFESPLAST has linked protein function with whole plant physiology. Novel insight has been gained into the early regulatory processes involved in Fe-S biogenesis. Project activities could ultimately be applied to enhance biomass production and agricultural productivity with important implications for global food security.