Systemic iron signaling via OPT3 influences reductive uptake and coumarin secretion in Arabidopsis

Iron (Fe) availability is often limited in soils, particularly under alkaline conditions, causing Fe-deficiency chlorosis and reduced plant growth. Strategy I plants respond through ferric chelate reductase (FCR)-dependent Fe reduction and the release of Fe-mobilizing compounds like coumarins, but how these responses are influenced by systemic Fe signaling and external pH remains unclear. Here, Arabidopsis thaliana Col-0, the systemic signaling mutant opt3-2 , and the coumarin-deficient mutant f6′h1-1 were examined using chlorophyll content, root FCR activity, and fluorescent coumarin accumulation as physiological and biochemical readouts. Plants were exposed to Fe-sufficient conditions, alkaline medium with low Fe bioavailability (navFe), or Fe-free medium at physiological pH. Under control conditions, all genotypes had similar chlorophyll levels and FCR activity, except opt3-2 which showed elevated basal FCR activity. Under navFe, opt3-2 maintained the highest chlorophyll content and a more sustained FCR response, while f6′h1-1 developed pronounced chlorosis despite strong FCR induction. FCR activity was generally higher under Fe-free conditions than under navFe, highlighting the influence of external pH on reductive Fe uptake. Fluorescent coumarins accumulated in Col-0 and opt3-2 but were absent in f6′h1-1 , with opt3-2 showing distinct temporal dynamics. Co-cultivation with opt3-2 partially alleviated Fe-deficiency symptoms in f6′h1-1 under navFe, improving chlorophyll and shoot fresh weight. Together, these findings show that external pH and OPT3-dependent systemic Fe signaling influence the balance between reductive Fe uptake and fluorescent coumarin-mediated Fe mobilization.