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Abstract Iron (Fe) uptake and translocation in plants are fine-tuned by complex mechanisms that are not yet fully understood. In Arabidopsis thaliana, local regulation of Fe homeostasis at the root level has been extensively studied and is better understood than the systemic shoot-to-root regulation. While the root system is solely a sink tissue that depends on photosynthates translocated from source tissues, the shoot system is a more complex tissue, where sink and source tissues occur synchronously. In this study, and to gain better insight into the Fe deficiency responses in leaves, we overexpressed Zinc/Iron-regulated transporter-like Protein (ZIP5), an Fe/Zn transporter, in phloem-loading cells (proSUC2::AtZIP5) and determined the timing of Fe deficiency responses in sink (young leaves and roots) and source tissues (leaves). Transgenic lines overexpressing ZIP5 in companion cells displayed increased sensitivity to Fe deficiency in root growth assays. Moreover, young leaves and roots (sink tissues) displayed either delayed or dampened transcriptional responses to Fe deficiency compared to wild-type (WT) plants. We also took advantage of the Arabidopsis mutant nas4x-1 to explore Fe transcriptional responses in the opposite scenario, where Fe is retained in the vasculature but in an unavailable and precipitated form. In contrast to proSUC2::AtZIP5 plants, nas4x-1 young leaves and roots displayed a robust and constitutive Fe deficiency response, while mature leaves showed a delayed and dampened Fe deficiency response compared to WT plants. Altogether, our data provide evidence suggesting that Fe sensing within leaves can also occur locally in a leaf-specific manner. 

Summary Mitochondria and chloroplasts are organelles with high iron demand that are particularly susceptible to iron‐induced oxidative stress. Despite the necessity of strict iron regulation in these organelles, much remains unknown about mitochondrial and chloroplast iron transport in plants. Here, we propose that Arabidopsis ferroportin 3 (FPN3) is an iron exporter that is dual‐targeted to mitochondria and chloroplasts.FPN3is expressed in shoots, regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots.fpn3mutants cannot grow as well as the wild type under iron‐deficient conditions and their shoot iron levels are lower compared with the wild type. Analyses of iron homeostasis gene expression infpn3mutants and inductively coupled plasma mass spectrometry (ICP‐MS) measurements show that iron levels in the mitochondria and chloroplasts are increased relative to the wild type, consistent with the proposed role of FPN3 as a mitochondrial/plastid iron exporter. In iron‐deficientfpn3mutants, abnormal mitochondrial ultrastructure was observed, whereas chloroplast ultrastructure was not affected, implying that FPN3 plays a critical role in the mitochondria. Overall, our study suggests that FPN3 is essential for optimal iron homeostasis. 

In order to discover sRNA that might function during iron deficiency stress, RNA was prepared from phloem exudates of Arabidopsis thaliana, and used for RNA-seq. Bioanalyzer results indicate that abundant RNA from phloem is small in size—less than 200 nt. Moreover, typical rRNA bands were not observed. Sequencing of eight independent phloem RNA samples indicated that tRNA-derived fragments, specifically 5′ tRFs and 5′ tRNA halves, are highly abundant in phloem sap, comprising about 46% of all reads. In addition, a set of miRNAs that are present in phloem sap was defined, and several miRNAs and sRNAs were identified that are differentially expressed during iron deficiency. 

Long non-coding RNAs (lncRNAs) are RNA molecules with functions independent of any protein-coding potential. A whole transcriptome (RNA-seq) study of Arabidopsis shoots under iron sufficient and deficient conditions was carried out to determine the genes that are iron-regulated in the shoots. We identified two previously unannotated transcripts on chromosome 1 that are significantly iron-regulated. We have called this iron-regulated lncRNA, CAN OF SPINACH ( COS ). cos mutants have altered iron levels in leaves and seeds. Despite the low iron levels in the leaves, cos mutants have higher chlorophyll levels than WT plants. Moreover, cos mutants have abnormal development during iron deficiency. Roots of cos mutants are longer than those of WT plants, when grown on iron deficient medium. In addition, cos mutant plants accumulate singlet oxygen during iron deficiency. The mechanism through which COS affects iron deficiency responses is unclear, but small regions of sequence similarity to several genes involved in iron deficiency responses occur in COS , and small RNAs from these regions have been detected. We hypothesize that COS is required for normal adaptation to iron deficiency conditions. 

Copper and iron are micronutrients but are toxic when they accumulate in cells in excess. Crosstalk between copper and iron homeostasis in Arabidopsis thaliana has been documented and includes iron accumulation under copper deficiency and vice versa. However, molecular components of this crosstalk are not well understood. Iron concentration in the phloem has been suggested to act systemically, negatively regulating iron uptake to the root. Consistently, systemic iron signaling is disrupted in A. thaliana mutants lacking the phloem companion cell-localized iron transporter, AtOPT3, and opt3 mutants hyperaccumulate iron. Here, we report that in addition to iron, AtOPT3 transports copper and mediates copper loading to the phloem for delivery from sources to sinks. As a result of this function, the opt3-3 mutant accumulates less copper in the phloem, roots, developing leaves and embryos compared to wild type, is sensitive to copper deficiency, and mounts transcriptional copper deficiency response. Because copper deficiency has been shown to stimulate iron accumulation, we propose that reduced copper concentration in the phloem of the opt3-3 mutant and its constitutive copper deficiency contribute to iron overaccumulation in its tissues. Our data assign new transport capabilities to AtOPT3 and increase understanding of copper - iron interactions and signaling.