Comparative transcriptome analysis of iron-overaccumulating Arabidopsis genotypes uncover novel putative regulators of systemic and retrograde signaling

Maintaining iron homeostasis requires a precise orchestration of cellular uptake, trafficking through mesophyll cells, translocation via xylem and phloem, and uptake of iron by roots adjusted to the demand of sink organs by systemic signals. Plants defective in the iron transporter OPT3 in the leaves behave as if they were constitutively iron deficient and accumulate toxic iron levels in their leaves. IRONMAN (IMA) genes encode peptides that trigger iron remobilization and uptake by inhibiting the ubiquitin ligase BRUTUS. Ectopic expression of IMA genes also causes iron accumulation. This study aims at bridging the knowledge gap of the mechanisms operating between OPT3-transported iron and the activation of IMA genes, and between phloem-localized IMAs and the activation of iron uptake genes in the epidermis. A small subset of genes was found to be shared among the three iron-accumulating genotypes and iron-deficient control plants. Root expression profiles were more strongly correlated that those of shoots, confirming a predominant function in the regulation of root processes by IMAs and OPT3. The largest overlap was observed between opt3-2 and wild-type plants. IMA1 Ox and IMA3 Ox lined showed a surprisingly small overlap except for the iron-related genes, suggesting distinct physiological functions. A small subset of genes was consistently differentially expressed in all the genotypes, including genes involved in root iron uptake and ROS detoxification. The small suite of differentially expressed genes common to all genotypes under investigation constitute potential novel regulators of cellular iron homeostasis. The study includes the RNAseq-based transcriptome profiling of the shoots and roots of the Fe-accumulating genotypes opt3-2, IMA1 Ox, IMA3 Ox and a transgenic control line expressing EYFP under the control of a 35s promoter. The plants were grown in vitro, on plates containing ES medium, under control and Fe-deficient conditions. Fe deficiency was achieved by transferring the plants for 3 days on medium without Fe and supplemented with 300 µM ferrozine.