Regulation of iron (Fe) acquisition and homeostasis is critical for plant survival. In Arabidopsis, Fe deficiency‐induced bHLH039 forms a complex with the master regulator FIT and activates it to upregulate Fe acquisition genes. FIT is partitioned between cytoplasm and nucleus, whereby active FIT accumulates more in the nucleus than inactive FIT. At the same time, there is so far no information on the subcellular localization of bHLH039 protein and how it is controlled. We report here that the bHLH039 localization pattern changes depending on the presence of FIT in the cell. When expressed in cells lacking FIT, bHLH039 localizes predominantly in the cytoplasm, including cytoplasmic foci in close proximity to the plasma membrane. The presence of FIT enhances the mobility of bHLH039 and redirects the protein toward primarily nuclear localization, abolishing its accumulation in cytoplasmic foci. This FIT‐dependent change in localization of bHLH039 found in transient fluorescent protein expression experiments was confirmed in both leaves and roots of Arabidopsis transgenic plants, stably expressing hemagglutinin‐tagged bHLH039 in wild‐type or fit mutant background. This posttranslational mechanism for intracellular partitioning of Fe‐responsive transcription factors suggests a signaling cascade that translates Fe sensing at the plasma membrane to nuclear accumulation of the transcriptional regulators.

Organisms require micronutrients, and Arabidopsis (Arabidopsis thaliana) IRON-REGULATED TRANSPORTER1 (IRT1) is essential for iron (Fe2+) acquisition into root cells. Uptake of reactive Fe2+ exposes cells to the risk of membrane lipid peroxidation. Surprisingly little is known about how this is avoided. IRT1 activity is controlled by an intracellular variable region (IRT1vr) that acts as a regulatory protein interaction platform. We found that IRT1vr interacted with peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN2 (PATL2). SEC14 proteins bind lipophilic substrates and transport or present them at the membrane. To date, no direct roles have been attributed to SEC14 proteins in Fe import. Here, PATL2 affected root Fe acquisition responses, interacted with ROS response proteins in roots, and alleviated root lipid peroxidation. PATL2 had high affinity in vitro for the major lipophilic antioxidant vitamin E compound α-tocopherol. Molecular dynamics simulations provided insight into energetic constraints and the orientation and stability of the PATL2-ligand interaction in atomic detail. Hence, this work highlights a compelling mechanism connecting vitamin E with root metal ion transport at the plasma membrane with the participation of an IRT1-interacting and α-tocopherol-binding SEC14 protein.

Reactive oxygen species play a central role in the regulation of plant responses to environmental stress. Under prolonged iron (Fe) deficiency, increased levels of hydrogen peroxide (H2O2) initiate signaling events, resulting in the attenuation of Fe acquisition through the inhibition of FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT). As this H2O2 increase occurs in a FIT-dependent manner, our aim was to understand the processes involved in maintaining H2O2 levels under prolonged Fe deficiency and the role of FIT in this process. We identified CAT2 gene, encoding one of the three Arabidopsis catalase isoforms, as regulated by FIT. CAT2 loss-of-function plants displayed severe susceptibility to Fe deficiency and greatly increased H2O2 levels in roots. Analysis of the Fe homeostasis transcriptional cascade revealed that H2O2 influences the gene expression of downstream regulators FIT, BHLHs of group Ib and POPEYE (PYE), however H2O2 did not affect their upstream regulators, such as BHLH104 and ILR3. Our data shows that FIT and CAT2 participate in a regulatory loop between H2O2 and prolonged Fe deficiency.

The endomembrane system is the central sorting machinery for membrane proteins where signals can be integrated to control plant nutritional responses. IRON-REGULATED TRANSPORTER1 (IRT1) is tightly controlled through trafficking between the endomembrane system and the plasma membrane to balance the demands for iron (Fe) and toxic effects that may arise from excess of this micronutrient in roots. Small GTPases of the Ras superfamily regulate signaling inputs in endomembrane trafficking, but their functions and activities are poorly known in plants. One plant endomembrane system-associated regulatory component is the small ARF-like GTPase TITAN5/ARL2/ARLC1/HALLIMASCH (hereafter termed TTN5). TTN5 is required during early embryo development but many open questions remain with regard to its physiological function during the later life cycle or in response to environmental cues. Here, we demonstrate that TTN5 interacts with IRT1vr, the large cytoplasmic variable region and protein-regulatory platform of IRT1. TTN5 also interacts with peripheral membrane proteins that are components of the IRT1 regulation machinery and can be involved in protein trafficking, like SNX1, the C2 domain protein EHB1/CAR6 and the SEC14-GOLD domain-containing PATL2. We show that heterozygous ttn5-1+/- plants have a Fe reduction phenotype. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at the plasma membrane and in endosomes/multivesicular bodies, where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. Hence, this work links IRT1 and iron nutritional regulation with a small GTPase. This opens up the possibility that iron nutrient levels are controlled via the cellular regulation of the endomembrane system.