The miR157-SPL-CNR module acts upstream of bHLH101 to negatively regulate iron deficiency responses in tomato

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(b HLH)TFs have been shown to contribute to Fe homeostasis,but the regulatory layers beyond b HLH TFs remain largely unclear.Here,we demonstrate that the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE(SPL)TF Sl SPL-CNR negatively regulates Fe-deficiency responses in tomato(Solanum lycopersicum)roots.Fe deficiency rapidly repressed the expression of Sl SPL-CNR,and Fe deficiency responses were intensified in two clustered regularly interspaced palindromic repeats(CRISPR)/CRISPRassociated protein 9-generated Sl SPL-CNR knockout lines compared to the wild-type.Comparative transcriptome analysis identified 47 Fe deficiencyresponsive genes the expression of which is negatively regulated by Sl SPL-CNR,one of which,Slb HLH101,helps regulate Fe uptake genes.Sl SPLCNR localizes the nucleus and interacts with the GTAC and BOX 4(ATTAAT)motifs in the Slb HLH101 promoter to repress its expression.Inhibition of Sl SPL-CNR expression in response to Fe deficiency was well correlated with the expression of the micro RNA Slymi R157.Slymi R157-overexpressing tomato lines displayed enhanced Fe deficiency responses,as did Sl SPL-CNR loss-of-function mutants.We propose that the Slymi R157-Sl SPL-CNR module represents a novel pathway that acts upstream of Slb HLH101 to regulate Fe homeostasis in tomato roots.