Fig. 6.

Autoregulation of NIGT1-clade genes. a Repression of NIGT1-clade gene promoters by NIGT1.1. A reporter plasmid that contained the LUC gene under the control of an NIGT1 promoter was co-transfected in protoplasts alongside the NIGT1.1 expression vector and the UBQ10-GUS reference plasmid. LUC activity was normalised with GUS activity. Data are means ± s.d. of three biological replicates. b Reduced expression of endogenous NIGT1.1 as well as NIGT1.2, NIGT1.3 and NIGT1.4 in NIGT1.1-OX lines. Ammonium-grown Arabidopsis Col and NIGT1.1-OX seedlings were treated with 10 mM KNO₃ for the indicated periods and used for RT-qPCR analysis. Values are normalised to UBQ10 expression levels, and means of biological triplicates with s.d. are shown. c Binding of NIGT1.1 to the NIGT1 gene promoters in vivo. ChIP analysis was performed with Col and NIGT1.1-OX seedlings. Two different promoter regions were amplified from immunoprecipitated DNA. Data are means of four biological replicates with s.d. **p < 0.01 by one-tailed t test compared with corresponding values obtained with Col seedlings. Positions of the conserved and nonconserved putative NIGT1-binding sites are shown by vertical red and orange lines, respectively. d Antagonistic regulation of NIGT1-clade gene promoters by NLP7 and NIGT1.1. Expression vectors for NLP7 and NIGT1.1 were co-transfected into protoplasts at different ratios, together with a reporter plasmid containing the LUC gene under the control of the NIGT1.1 or NIGT1.3 promoter. Transfected protoplasts were incubated in the presence of 10 mM KCl or KNO₃. LUC activity was normalised with GUS activity, and relative LUC activities are means ± s.d. of three biological replicates