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.

Plant vacuoles serve as the primary intracellular compartments for phosphorus (P) storage and play a central role to maintain P homeostasis. The Oryza sativa (rice) genome contains three genes that encode SPX (SYG1/PHO81/XPR1)-MFS (Major Facility Superfamily) proteins (OsSPX-MFS1, 2, 3). OsSPX-MFS1 and OsSPX-MFS3 were shown previously to have vacuolar phosphate (Pi) transporter activities, but the physiological role of the three transporters under varying P conditions and under field grown conditions for a crop plant is not known. To address this knowledge gap, we generated single, double, and triple mutants (7 mutants with at least two lines of each) for the three rice Os SPX-MFS genes. All the mutants except osspx-mfs2 display lower vacuolar Pi concentrations and all Os SPX-MFSs overexpression plant lines display higher Pi accumulation, demonstrating that all three OsSPX-MFSs are vacuolar Pi influx transporters. OsSPX-MFS3 plays the dominant role based on the phenotypes of three single mutants in terms of growth, vacuolar and tissue Pi concentrations. OsSPX-MFS2 is the weakest and only functions as vacuole Pi sequestration under osspx-mfs1/3 background. The vacuolar Pi sequestration was severely impaired in osspx-mfs1/ 3 and osspx-mfs1/2/ 3, which led to Pi toxicity and subsequently increased Pi allocation to aerial organs. High Pi in the panicle result in necrotic symptoms on husks and impaired panicle and grain development in osspx-mfs1/ 3 and osspx-mfs1/2/ 3 mutant lines. The mutation in the weak vacuolar Pi transporter OsSPX-MFS2 resulted more stable yield compared to the wildtype under low P field conditions. The results suggest that alteration of vacuolar Pi sequestration may be a novel effective strategy to improve rice (crop) tolerance to low phosphorus field conditions and maintain yield.