Abstract:

Iron (Fe) homeostasis is critical for plant growth, development, and stress responses. Fe levels are tightly controlled by intricate regulatory networks in which transcription factors (TFs) play a central role. A series of basic helix-loop-helix (bHLH) TFs have been shown to contribute to Fe homeostasis, but the regulatory layers beyond bHLH TFs remain largely unclear. Here, we demonstrate that the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) TF SlSPL-CNR negatively regulates Fe-deficiency responses in tomato (Solanum lycopersicum) roots. Fe deficiency rapidly repressed the expression of SlSPL-CNR, and Fe deficiency responses were intensified in two clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-generated SlSPL-CNR knock-out lines compared to the wild-type. Comparative transcriptome analysis identified 47 Fe deficiency-responsive genes the expression of which is negatively regulated by SlSPL-CNR, one of which, SlbHLH101, helps regulate Fe uptake genes. SlSPL-CNR localizes the nucleus and interacts with the GTAC and BOX 4 (ATTAAT) motifs in the SlbHLH101 promoter to repress its expression. Inhibition of SlSPL-CNR expression in response to Fe deficiency was well correlated with the expression of the microRNA SlymiR157. SlymiR157-overexpressing tomato lines displayed enhanced Fe deficiency responses, as did SlSPL-CNR loss-of-function mutants. We propose that the SlymiR157-SlSPL-CNR module represents a novel pathway that acts upstream of SlbHLH101 to regulate Fe homeostasis in tomato roots.