The bHLH Transcription Factor PhbHLH121 Regulates Response to Iron Deficiency in Petunia hybrida

Iron (Fe) is an essential micronutrient for plants. Due to the low Fe bioavailability in cultivated soils, Fe deficiency is a widespread agricultural problem. In this study, we present the functional characterization of a petunia (<i>Petunia hybrida</i>) basic-helix-loop-helix transcription factor PhbHLH121 in response to Fe shortage. Real-time PCR revealed that the expression of <i>PhbHLH121</i> in petunia roots and shoots was downregulated under Fe-limited conditions. CRISPR/Cas9-edited <i>phbhlh121</i> mutant plants were generated to investigate the functions of PhbHLH121 in petunia. Loss-of-function of PhbHLH121 enhanced petunia tolerance to Fe deficiency. Further investigations revealed that the expression level of several structural genes involved in Fe uptake in petunia, such as <i>IRT1</i> and <i>FRO2</i>, was higher in <i>phbhlh121</i> mutants compared to that in wild-type under Fe-limited conditions, and the expression level of several genes involved in Fe storage and Fe transport, such as <i>VTL2</i>, <i>FERs</i> and <i>ZIF1</i>, was lower in <i>phbhlh121</i> mutants compared to that in wild-type under Fe-deficient conditions. Yeast one-hybrid assays revealed that PhbHLH121 binds to the G-box element in the promoter of genes involved in Fe homeostasis. Yeast two-hybrid assays revealed that PhbHLH121 interacts with petunia bHLH IVc proteins. Taken together, PhbHLH121 plays an important role in the Fe deficiency response in petunia.