CITF1 interacts with FIT and regulates copper–iron crosstalk in Arabidopsis

Abstract Iron (Fe) and copper (Cu) are essential yet potentially toxic metals with interconnected metabolic pathways; however, the mechanisms underlying Fe–Cu crosstalk remain poorly defined. Here, we show that CITF1 (COPPER DEFICIENCY INDUCED TRANSCRIPTION FACTOR 1), a Cu homeostasis regulator in Arabidopsis thaliana, physically interacts with FIT (FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR), the central Fe homeostasis regulator, forming a nutrient-responsive transcriptional module. Under Cu deficiency, the CITF1–FIT complex accumulates and promotes expression of the Cu uptake genes COPT2 (COPPER TRANSPORTER 2), FRO4 (FERRIC REDUCTION OXIDASE 4), and FRO5 (FERRIC REDUCTION OXIDASE 5). Proteasome-dependent degradation regulates CITF1 and FIT stability, with Cu deficiency delaying their turnover in a CITF1-dependent manner. Under Fe deficiency, CITF1 expression is downregulated, allowing FIT to interact with bHLH38/39/100/101 partners and activate Fe uptake genes, as CITF1 disrupts these interactions. Thus, CITF1 negatively regulates Fe acquisition. Consistent with this, citf1-1 and citf1-2 mutants show reduced sensitivity to Fe deficiency. Under Cu deficiency, the citf1-2 and fit-2 mutants have additive effects and under Fe deficiency, the double mutant shows partial suppression of the fit-2 slow growth phenotype, supporting the positive and negative roles of CITF1 in Cu and Fe homeostasis, respectively. Complete loss of CITF1 function in the homozygous citf1-1 fit-2 double mutant causes embryo lethality, revealing roles for CITF1 and FIT in embryo development. These findings establish CITF1 as a nutrient-responsive regulator of Cu/Fe crosstalk, functioning through interactions with FIT to prioritize Cu or Fe acquisition and balance micronutrient homeostasis.