The iron-regulated control network of nutrient uptake in plants

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Iron (Fe) and zinc (Zn) are essential micronutrients for plants, which are the main entry point of these minerals into the food chain. Therefore, an understanding of how plants regulate Fe and Zn uptake is vitally important to maximise crop yields and produce more nutritious plant-based foods. Previously, we have functionally characterised two partially redundant genes that occupy a central position in Fe-regulated gene networks: BTSL1 and BTSL2 which encode hemerythrin E3 ubiquitin ligases distantly related to a mammalian regulator of Fe homeostasis. We showed that the BTSL proteins negatively regulate the Fe deficiency response, by targeting a key transcription factor in Fe uptake, named FIT, for degradation (Rodriguez-Celma et al. 2019 PNAS). As a consequence, btsl mutants accumulate more Fe than wild-type plants. The btsl mutants are also tolerant to Zn excess, but the mechanisms for this is not understood. Despite significant progress in unravelling the biological function of the BTSL proteins, how they respond to the Fe and Zn concentration in the cell is still an open question. Here we propose to elucidate the molecular mechanism by which metal binding to the N-terminal hemerythrin domain regulates the activity of the C-terminal E3 ligase domain of BTSL2, which is the dominant isoform of the two paralogues. First, we will identify additional protein substrates of BTLS2 to enhance our toolkit for studying E3 ligase activity in vivo. Second, we will study how binding of Fe and Zn affects folding of the N-terminus of BTSL2 in vitro, and if oxygen and reactive oxygen species (ROS) can interact with the di-Fe sites. Third, we will test how different concentration of Fe, Zn and ROS affect the stability of BTSL2 in plant tissue. Information from these first 3 sets of experiments will be integrated into the design of mutagenesis studies to test how BTS and BTSL respond to varying Fe and Zn concentrations in different parts of plants.