A SQUAMOSA PROMOTER BINDING PROTEIN, LeSPL-CNR targeted by SlymiR157 negatively regulates Fe deficiency response in tomato roots

Iron (Fe) homeostasis is critical for plant growth, development and stress response, which is tightly controlled by intricated regulatory networks in which transcription factors (TFs) play a central role. Whilst a series of basic helix-loop-helix (bHLH) TFs have been functionally characterized to contribute to Fe homeostasis, regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that a SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF, LeSPL-CNR, negatively regulates Fe deficiency responses in tomato roots. Fe deficiency rapidly repressed the expression of LeSPL-CNR. However, Fe-deficiency-responses were intensified in two LeSPL-CNR CRISPR/Cas9 gene editing transgenic lines compared with wild-type plants. By comparative transcriptome analyses, we identified 47 Fe-deficiency responsive genes including SlbHLH101 whose expression were negatively regulated by LeSPL-CNR. LeSPL-CNR is localized to nucleus and yeast one-hybrid and luciferase reporter assays revealed that LeSPL-CNR could interact with GTAC motif and BOX4 motif of SlbHLH101 promoter to repress its expression. Moreover, Fe deficiency-induced expression inhibition of LeSPL-CNR is correlated well with SlymiR157 expression induction. Additionally, transgenic tomato overexpressing SlymiR157 displayed improved Fe deficiency responses similar to that of LeSPL-CNR loss-of-function mutants. Altogether, we propose that miR157-LeSPL-CNR module provided an additional pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.