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. FPN3 is expressed in shoots, regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots. fpn3 mutants 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 in fpn3 mutants 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-deficient fpn3 mutants, 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.

Iron is an essential micronutrient for nearly all organisms, but excessive iron can lead to the formation of cytotoxic reactive oxygen species. Therefore, iron acquisition and homeostasis must be tightly regulated. Plants have evolved complex mechanisms to optimize their use of iron, which is one of the most limiting nutrients in the soil. In particular, transcriptional regulation is vital for regulating iron in plants, and much work has revealed the role of transcription factors on this front. Our study adds novel insights to the transcriptional regulation of iron homeostasis in plants by showing that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency. We provide evidence that FIT-dependent iron acquisition genes, IRT1 and FRO2 , as well as FIT itself are direct targets of PRC2-mediated H3K27me3. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, induction of FIT , FRO2 , IRT1 , and other FIT-regulated genes in roots is significantly higher under iron deficient conditions than in wild type. Furthermore, we observe that clf mutants are more tolerant to iron deficiency than wild type, indicating that gene expression levels appear to be limiting the plants ability to access iron. We propose that H3K27me3 attenuates the induction of FIT-target genes under iron deficiency and hypothesize that this may serve as a mechanism to restrict the maximum level of induction of iron acquisition genes to prevent iron overload.

Microglia, the resident macrophages of the brain, play a key role in the pathogenesis of Human Immunodeficiency Virus (HIV)-associated neurocognitive disorders (HAND) due to their productive infection by HIV. This results in the release of neurotoxic viral proteins and pro-inflammatory compounds which negatively affect the functionality of surrounding neurons. Because models of HIV infection within the brain are limited, we aimed to create a novel microglia cell line with an integrated HIV-provirus capable of recreating several hallmarks of HIV infection. We utilized CRISPR/Cas9 gene editing technology and integrated a modified HIV provirus into CHME-5 immortalized microglia to create HIV-NanoLuc CHME-5. In the modified provirus, the Gag-Pol region is replaced with the coding region for Nanoluciferase (NanoLuc), which allows for the rapid assay of HIV LTR activity using a luminescent substrate, while still containing the necessary genetic material to produce established neurotoxic viral proteins (e.g. tat, nef, gp120). We confirmed that HIV-NanoLuc CHME-5 microglia express NanoLuc, along with the HIV viral protein Nef. We subsequently exposed these cells to a battery of experiments to modulate the activity of the provirus. Proviral activity was enhanced by treating the cells with pro-inflammatory factors LPS and TNF-α and by overexpressing the viral regulatory protein Tat. Conversely, genetic modification of the TLR-4 gene by CRISPR/Cas9 reduced LPS-mediated proviral activation, and pharmacological application of NF-κB inhibitor sulfasalazine similarly diminished proviral activity. Overall these data suggest that HIV-NanoLuc CHME-5 may be a useful tool in the study of HIV-mediated neuropathology and proviral regulation.

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