Roles of the OsbHLH156-OsIRO2 transcription factor complex in manganese, copper and zinc transport in rice

Iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) are essential micronutrients that are necessary for plant growth and development, but can be toxic at supra-optimal levels. Plants have evolved a complex homeostasis network that includes uptake, transport, and storage of these metals. Previously, we reported that the OsbHLH156-OsIRO2 transcription factor (TF) complex is activated under Fe deficient condition and acts as a central regulator on Strategy II Fe acquisition. In this study, the role of the TF complex on Mn, Cu and Zn uptake was evaluated. Fe deficiency caused significant increased Mn, Cu and Zn concentrations in shoot. The increase of these divalent metal concentrations was significantly suppressed in osbhlh156 and osiro2 mutants, suggesting that the TF complex plays roles on Mn, Cu and Zn uptake and transport. RNA-sequencing assay showed that the genes associated with Mn, Cu, and Zn uptake and transport were significantly suppressed in the osbhlh156 and osiro2 mutants. Transcriptional activation assays demonstrated that the TF complex could directly bind to the promoters of OsIRT1 , OsYSL15 , OsNRAMP6 , OsHMA2 , OsCOPT1/7 and OsZIP5/9/10 and activate their expression. In addition, the TF complex is required to activate the expression of NA and 2’deoxymugineic acid (DMA) synthesis genes, which in turn facilitate the uptake and transport of Mn, Cu and Zn. Furthermore, OsbHLH156 and OsIRO2 promote Cu accumulation to partially restore the Fe-deficiency symptom. Taken together, the OsbHLH156-OsIRO2 TF complex functions as a core regulator not only in Fe homeostasis, but also in Mn, Cu and Zn accumulation.