The BRUTUS-LIKE E3 ligases BTSL1 and BTSL2 fine-tune transcriptional responses to balance Fe and Zn homeostasis

The mineral micronutrients zinc (Zn) and iron (Fe) are essential for plant growth and human nutrition, but interactions between the homeostatic networks of these two elements are not fully understood. Here we show that loss-of-function of BTSL1 and BTSL2 , which encode partially redundant E3 ubiquitin ligases that negatively regulate Fe uptake, confers tolerance to Zn excess in Arabidopsis thaliana . Double btsl1 btsl2 mutant seedlings grown on high Zn medium accumulated similar amounts of Zn in roots and shoots as the wild type, but suppressed the accumulation of excess Fe in roots. RNA-seq analysis showed that roots of mutant seedlings had relatively higher expression of genes involved in Fe uptake ( IRT1, FRO2, NAS ) and in Zn storage ( MTP3, ZIF1 ). Surprisingly, mutant shoots did not show the transcriptional Fe-deficiency response which is normally induced by Zn excess. Split-root experiments suggested that within roots the BTSL proteins act locally and downstream of systemic Fe deficiency signals. Together, our data show that constitutive low-level induction of the Fe-deficiency response protects btsl1 btsl2 mutants from Zn toxicity. We propose that BTSL protein function is disadvantageous in situations of external Zn and Fe imbalances, and formulate a general model for Zn-Fe interactions in plants. Highlight Mutation of two E3 ligases that suppress iron uptake in roots also confers tolerance to zinc toxicity, identifying a regulatory point of interaction between iron and zinc homeostasis. ### Competing Interest Statement The authors have declared no competing interest. * bHLH : basic helix-loop-helix BTS : BRUTUS BTSL : BRUTUS-LIKE FBP : FIT-Binding Protein FCR : Ferric Chelate Reductase Fe : iron FIT : FER-like Iron deficiency induced Transcription factor FRO2 : Ferric Reduction Oxidase 2 FW : fresh weight GSH : glutathione HBED : N,N′-bis(2-hydroxyphenyl)ethylenediamine-N,N′-diacetic acid HMA : Heavy Metal ATPase IRT1 : Iron-Regulated Transporter1 Mn : manganese MTP : Metal Tolerance Protein NA : nicotianamine NAS : Nicotianamine Synthase ZIF : Zinc-Induced Facilitator Zn : zinc