Fig. 3: BRM and STOP1 antagonistically regulate NRT1.1 expression.
a Schematic representation of the effector and reporter constructs used in the transient dual-luciferase assays. All constructs contain a NOS terminator (not shown). Different NRT1.1 genomic fragments were inserted upstream of the CaMV 35S minimal promoter (Mini; −46/0). b, c Effect of BRM on STOP1 binding to the NRT1.1 genomic regions assessed via transient dual-luciferase assays. The empty vector (EV, pCambia1300) was used as a negative control (set to 1). Effector and reporter constructs were co-transformed into N. benthamiana leaves for 48 h, after which the ratio of firefly to renilla luciferase activity was measured (n = 4 biological replicates). d ChIP-qPCR assay performed in Col-0 and brm-3 protoplasts transiently expressing HBT:STOP1-GFP or the control HBT:GFP construct. Immunoprecipitation was performed using an anti-GFP antibody (n = 3 biological replicates). e Genome browser snapshot showing BRM binding sites at the NRT1.1 locus. The BRM ChIP-seq data shown was downloaded from GEO under accession no. GSM4233035 and the ATAC-seq data were downloaded from GEO under accession no. GSM5799538. f ChIP-qPCR assay using 10-day-old Col-0 and pBRM:BRM-FLAG/brm-3 seedlings, with immunoprecipitation using an anti-FLAG antibody. Fold enrichment represents IP efficiency in transgenic plants normalized to that in Col-0 (n = 3 biological replicates). g FAIRE-qPCR analysis of chromatin accessibility in the P1-1 and P2-1 regions of NRT1.1, relative to the UBQ10 coding sequence. Whole 10-day-old Arabidopsis seedlings were used. Relative amplicon abundance represents signal in the mutants normalized to Col-0 (n = 3 biological replicates). h, i Comparison of root growth between Col-0 and axe1-5 mutants. The 3-day-old Arabidopsis seedlings were transferred to a neutral- or low-pH medium for 2 days. Scale bar: 1 cm. Centerlines in the boxplots show the medians, box limits indicate the 25th and 75th percentile, and whiskers indicate the minimum and maximum values (n = 20 seedlings). j Transcription of NRT1.1 in roots. The 6-day-old Arabidopsis seedlings were transferred to a neutral- or low-pH medium for 1 days (n = 3 biological replicates). In (c, d, f, g, j), data are shown as mean ± SD. Significant differences were determined by two-tailed Student’s t-test in (c, d, f, i, j), or two-way ANOVA in (c, g). Exact p values are provided in the Source Data file. All experiments were repeated at least three times independently, with similar results.
