The assembly of iron-sulphur proteins in germinating seedlings

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Seeds and grains store iron that is rapidly mobilized upon germination for incorporation into an array of essential enzymes. As catalytic sites, iron-sulphur (Fe-S) clusters are much more common in plants than haem and other Fe cofactors, but much less is known about their biosynthesis. The assembly of Fe-S clusters in plants takes place in three cell compartments, the mitochondria, plastids and cytosol. During germination, high activities of mitochondrial electron transfer complexes and a transient burst in cytosolic aconitase depend on Fe-S clusters and their rapid assembly. This is followed by expression of the Fe-S rich photosynthetic machinery from day 5. In this proposal, we will focus on the as yet obscure cytosolic pathway, in finding the source of iron, and how S and Fe are delivered and assembled on the cytosolic scaffold protein NBP35. Following germination from day 1 to 10, we will carry out a time course of subcellular Fe pools, and link this to changes in expression of abundant Fe-S enzymes and cofactor assembly genes. Genome-wide co-expression analysis will be carried out to identify novel candidates involved in the assembly process. S and Fe delivery to NBP35 will be studied by monitoring its conformational changes in vivo and in vitro, while critical amino acids for S and Fe binding to NBP35 will be studied by point mutagenesis in vitro. To identify interactions partners of NBP35 we will carry out co-immunoprecipitation and yeast-2-hybrid studies. The various candidate genes for cytosolic Fe-S assembly will then be subjected to functional characterization studies using Arabidopsis mutants.
The outcome of this study is basic knowledge on the transport and utilization of Fe in germinating seedlings, which is important for crop yield, human nutrition and plant biotechnology, for example to express transgenic Fe-S enzymes such as nitrogenase.

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