Molecular Mechanism by Which the BrFIT2–BrbHLH–BrIRT1 Module Synergistically Regulates Iron Absorption in <i>Brassica rapa</i> ssp. <i>Pekinensis</i>

Iron is essential for plant photosynthesis and human health, yet Brassica rapa ssp. pekinensis exhibits significantly lower iron content compared to other leafy vegetables, and its iron uptake mechanisms are not well understood. This study elucidates the molecular mechanisms governing iron uptake by investigating the roles of the iron-regulated gene BrFIT2 (Bra011972) and the iron transport protein gene BrIRT1 (Bra013419) through yeast two-hybrid, bimolecular fluorescence complementation, genetic transformation and physiological-biochemical analyses, etc. Key findings include: BrFIT2 encodes a nuclear transcription factor containing an HLH domain, with its expression in roots upregulated 6.9-fold under iron-deficient conditions. Overexpression of BrFIT2 increased iron content in leaves and roots by 1.42- and 6.17-fold, respectively, whereas silencing BrFIT2 reduced leaf iron uptake by 60%-80%. Furthermore, BrFIT2 interacts with BrbHLH38/100 to form nuclear complexes that activate the expression of the iron transporter gene BrIRT1. Overexpression of BrIRT1 resulted in root iron levels 16.29-fold higher than those in wild-type plants. This study is the first to delineate the BrFIT2-BrbHLH-BrIRT1 regulatory cascade in Brassica rapa, challenging the conventional model of direct FIT-target gene interactions.