Fig. 1

Repression of the NRT2.1 promoter by NIGT1.1. a Structure of the NRT2.1 promoter. Consensus sequences for NIGT1 binding (5′-GAATC-3′) and LBD binding (5′-GCGGCG-3′) are indicated by red and green bars, respectively. White and black boxes indicate the 5′ untranslated and coding regions, respectively. Probe DNAs used in EMSA (c, d) are shown below the promoter structure. To disrupt the NIGT1-binding sequences, mutated probes contained nucleotide substitutions, indicated by X. b Activation of the NRT2.1 promoter by nitrate and NLP7 and repression by NIGT1.1 in protoplasts. Protoplasts co-transfected with the NLP7, NIGT1.1 or LBD37 expression vector or the empty vector (none) together with the reporter plasmid containing the LUC gene fused to the NRT2.1 promoter were incubated in the presence of 1 mM KCl or KNO₃. In b and e, LUC activity was normalised with GUS activity from the reference UBQ10-GUS plasmid and data are means ± s.d. of three biological replicates. c, d EMSA with recombinant NIGT1.1 protein and DNA probes from the NRT2.1 promoter. Red arrowheads indicate positions of protein–DNA complexes caused by binding of NIGT1.1 to probe DNA. The OsNIGT1 probe served as a positive control. e Effects of disruption of the NIGT1-binding sites on the NRT2.1 promoter activity in protoplasts. Protoplasts co-transfected with the LUC gene fused to the wild-type or mutant NRT2.1 promoter and the NLP7 expression vector or an empty vector were incubated in the presence of 1 mM KCl or KNO₃. X indicates disrupted NIGT1 sites. f ChIP analysis of the NRT2.1 promoter using Col and NIGT1.1-OX seedlings. Four regions were amplified by PCR with immunoprecipitated DNA. Data are means of four biological replicates with s.d. **p < 0.01 by one-tailed t test. g Effects of disruption of the NIGT1-binding sites on NRT2.1 promoter activity in the presence of abundant N. Five seedlings of transgenic lines harbouring the LUC gene fused to the wild-type [NRT2.1p(WT)-LUC] or the mutated [(mut1+2)-LUC] NRT2.1 promoter were incubated with (+) or without (−) 10 mM NH₄NO₃ for 24 h. Images of LUC activity in vivo and bright-field images were captured in two independent transgenic lines. Bar, 2 cm