Highlights d Iron-regulated and calcium-dependent protein kinase CIPK11 interacts with FIT d CIPK11 and calcium sensors CBL1/9 promote FIT-dependent Fe deficiency responses d FIT is phosphorylated in plants, and CIPK11 phosphorylates FIT at Ser272 d Mutation at Ser272 modulates FIT activity, affecting seed iron content

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.

Summary The key basic helix–loop–helix (bHLH) transcription factor in iron (Fe) uptake, FER‐LIKE IRON DEFICIENCY‐INDUCED TRANSCRIPTION FACTOR (FIT), is controlled by multiple signaling pathways, important to adjust Fe acquisition to growth and environmental constraints. FIT protein exists in active and inactive protein pools, and phosphorylation of serine Ser272 in the C‐terminus, a regulatory domain of FIT, provides a trigger for FIT activation. Here, we use phospho‐mutant activity assays and study phospho‐mimicking and phospho‐dead mutations of three additional predicted phosphorylation sites, namely at Ser221 and at tyrosines Tyr238 and Tyr278, besides Ser 272. Phospho‐mutations at these sites affect FIT activities in yeast, plant, and mammalian cells. The diverse array of cellular phenotypes is seen at the level of cellular localization, nuclear mobility, homodimerization, and dimerization with the FIT‐activating partner bHLH039, promoter transactivation, and protein stability. Phospho‐mimicking Tyr mutations of FIT disturb fit mutant plant complementation. Taken together, we provide evidence that FIT is activated through Ser and deactivated through Tyr site phosphorylation. We therefore propose that FIT activity is regulated by alternative phosphorylation pathways.

Several nuclear proteins undergo condensation. The question remains often whether this property is coupled to a functional aspect of the protein in the nucleus. The basic helix-loop-helix (bHLH) FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) integrates internal and external signals to control the amount of iron that is acquired in accordance with growth. The previously described C-terminal Ser271/272 allows FIT to form active complexes with subgroup Ib bHLH factors such as bHLH039. FIT has lower nuclear mobility than mutant FITmSS271AA, but this behavior has remained mechanistically and functionally obscure. Here, we show that FIT undergoes a light-inducible subnuclear partitioning into nuclear condensates that we termed FIT nuclear bodies (NBs). The characteristics of FIT NBs could be examined using a standardized FIT NB analysis procedure coupled with different types of quantitative and qualitative microscopy-based approaches. We found that FIT condensates were likely formed by liquid-liquid phase separation. FIT accumulated preferentially in FIT NBs versus nucleoplasm when engaged in protein complexes with itself and with bHLH039. FITmSS271AA, instead, localized to NBs with different dynamics. FIT colocalized with splicing and light signaling NB markers. Hence, the inducible highly dynamic FIT condensates link active transcription factor complexes with posttranscriptional regulation processes. Active transcription factor complexes for Fe signaling may form as nuclear condensates to integrate environmental signals and Fe nutrition.