Identification of miRNAs Responsive to a Defined Period of Iron Deficiency and Resupply in Arabidopsis thaliana

Iron (Fe), as one of the essential micronutrients for plants, plays a pivotal role in regulating growth and development through homeostatic balance. Fe deficiency is a common agricultural stress that causes visible leaf chlorosis and impairs plant growth. In this study, Arabidopsis thaliana seedlings grown under Fe deficiency for 4 days were subjected to 6 h Fe resupply via foliar spray or root supply, followed by measurements of chlorophyll fluorescence and metal ion contents in leaves and roots. Fe deficiency significantly reduced Fe levels and the maximum quantum yield of fluorescence (Fv/Fm), while increasing copper (Cu) accumulation in roots. Zinc (Zn) and manganese (Mn) levels were also altered, depending on tissue type. Fe resupply restored Fv/Fm, increased Mn levels, and rebalanced micronutrient content. MicroRNA (miRNA) mediates adaptation to Fe deficiency via post-transcriptional regulation in plants. However, the involved regulatory networks of miRNAs under stress conditions during Fe resupply following deficiency remain poorly understood. These physiological changes prompted us to explore the underlying regulatory networks using miRNA-seq and mRNA-seq. The bioinformatics analysis identified differentially expressed miRNAs responsive to Fe stress, with the Fe-deficiency-specific cis-element IDE1 characterized in their promoter regions. By integrating miRNA-seq and mRNA-seq datasets, we constructed a regulatory network and identified 13 miRNAs harboring IDE1 motifs alongside their functional target genes. Three critical Fe homeostasis modules were proposed—miR396b-LSU2, miR401-HEMA1, and miR169b-NF-YA2—that link Fe homeostasis to chlorophyll synthesis, sulfur (S) responses, and developmental signaling. This study integrates physiological phenotyping with transcriptomic insights to provide a comprehensive view of Fe deficiency and recovery in Arabidopsis.