Fig. 7: OsRBOH-D is a direct target gene of OsPHR2.

a The relative expression levels of OsRBOH-D and OsRBOH-H in wild type XS134, OsPHR2 overexpression plant (PHR2-OE1) and phr2 mutant determined by RT-qPCR. OsACTIN1 was used as an internal control. b A schematic diagram of the OsRBOH-D genomic region. P represents the DNA fragments amplified in the ChIP-qPCR assays. Red short lines indicate the positions of the P1BS motif. c, d Dual-luciferase (LUC) reporter gene assays to examine the role of OsPHR2 on OsRBOH-D expression in leaves of N. benthamiana plants pretreated with 2 mM H₂O₂ or LP for 2 days. GFP, OsPHR2-GFP, C140S-GFP, or C377S-GFP were used as an effector, and LUC driven by the OsRBOH-D promoter was used as a reporter. The LUC activity of OsRBOH-Dpro::LUC + GFP under mock conditions was set to 1. e Enrichment of the OsRBOH-D promoter fragment indicated in ChIP assays. Chromatin from 7-day-old GFP/phr2, GFP/phr2rboh-DH, OsPHR2-GFP/phr2, OsPHR2-GFP/phr2rboh-DH, C140S-GFP/phr2, and C377S-GFP/phr2 plants treated with 2 mM H₂O₂ or LP for an additional 2 days were immunoprecipitated using the anti-GFP antibody and then used for qPCR. f The binding affinity of MBP-OsPHR2 to OsRBOH-D promoter (left) and the effects of H₂O₂ on the binding affinity of MBP-OsPHR2 or MBP-C377S to OsRBOH-D promoter (right) determined by EMSA assay. g Mutations in the binding motif of OsRBOH-D promoter (RBOH-D-mP) reduce the binding intensity of OsPHR2 to the OsRBOH-D promoter determined by EMSA assay. h Working model for the mechanism underlying LP-triggered H₂O₂ accumulation and its effect on OsPHR2 to enhance Pi-starvation responses in rice. Under LP stress, OsRBOH-D/H are rapidly induced to facilitate H₂O₂ production to oxidize OsPHR2 at Cys377, which promotes the Cys377/Cys777-mediated oligomerization of OsPHR2 to enhance its nuclear translocation and DNA binding ability to promoters of PSI genes, including OsRBOH-D, thus activating their expression to improve rice LP adaptation. The EMSA analysis was repeated three times with similar results for f and g. Data are means ± SD (n  =  3 replicates for a, d, and e). The P-value was generated from a two-sided Student’s t test for a. Different letters above the bars denote significant differences (P  <  0.05) according to a two-sided Duncan’s multiple range test for d and e. Source data are provided in the Source Data file.