ScycA1;1, Oscdc2Os-3, or OsGRF4 were amplified from NJ6, after which subcloned into a pUC19 vector containing the firefly LUC reporter gene driven by the 35S minimal TATA box and 5 AL4 binding components, as a result creating reporter plasmids containing distinct promoters fused to LUC. The fulllength OsGRF4 cDNA was amplified and fused to sequence encoding GAL4BD, thus creating the effector plasmid pRTBD-OsGRF4. Transient transactivation assays were performed using rice protoplasts as described elsewhere47. The Dual-Luciferase Reporter Assay Technique (Promega, E1960) was applied to perform the luciferase activity assay, together with the Renilla LUC gene as an internal handle. Relevant primer sequences are given in Supplementary Info Table six.Determination of plant C and N concentration Samples from many plant organs had been dried in an oven at 80 for 72 hours. Following tissue homogenisation, C and N concentrations had been determined applying an elemental analyser (IsoPrime100; Elementar). All experiments were performed with no less than three (S)-Flurbiprofen Inhibitor replicates.15Nuptake evaluation Following development in hydroponic culture for four weeks, rice root 15NO3- and 15NH4+ influx measurements were as described elesewhere48,49. Roots and shoots have been separated andNature. Author manuscript; readily available in PMC 2019 February 15.Li et al.Pagestored at -70 just before freeze drying. Roots and shoots were dried overnight at 80 , and the 15N content was measured applying the Isoprime 100 (Elementar, Germany). Determination of glutamine synthase and nitrate reductase activities Glutamine synthase and nitrate reductase activities had been respectively determined using the Glutamine Synthetase Kit (Solarbio LIFE SCIENCES, BC0910) along with the Nitrate Reductase Kit (Solarbio LIFE SCIENCES, BC0080) following the manufacturer’s directions.Europe PMC Funders Author Manuscripts Europe PMC Funders Author ManuscriptsExtended DataExtended Information Figure 1. Allelic variation at the OsGRF4 locus affects OsGRF4 mRNA abundance and root 15NH4+ uptake.a, Positional ML-180 Cancer cloning indicates the equivalence of OsGRF4 with qNGR2 (N-mediated development response two). Successive maps show progressive narrowing of concentrate of qNGR2 (red dot,Nature. Author manuscript; accessible in PMC 2019 February 15.Li et al.Pageusing recombination break points and linked DNA markers) to an two.7-kbp area on chromosome 2 flanked by molecular markers L17 and L18 and overlapping candidate gene LOC_Os02g47280 (also referred to as OsGRF4). The start off ATG (nucleotide 1) and close TGA (nucleotide 3385) of OsGRF4 are shown, with each other with protein-encoding DNA sequence (CDS, thick black bars). The target web page for OsmiR396 is indicated by an . The structure of a CRISPRCas9 generated osgrf4 mutant 91-bp deletion allele spanning parts of exon 1 and intron 1 is shown. b, 15NH4+ uptake rates of roots of BC2F2 progeny (derived from a NJ6 NM73 cross) homozygous or heterozygous for OsGRF4NGR2 or OsGRF4ngr2 grown in high N supply (1.25 mM NH4NO3). Information shown as imply s.e.m. (n = 9). Distinctive letters denote substantial variations (P 0.05, Duncan’s many range test). c, OsGRF4 mRNA abundance in plants (genotypes as shown) relative to the abundance in NJ6 (set to one). Information shown as mean s.e.m. (n = three). Diverse letters denote substantial variations (P 0.05, Duncan’s multiple range test). d, Organic varietal OsGRF4 allelic variation. Nucleotide position relative to the OsGRF4 begin ATG is shown inside a. SNPs shared involving varieties NM73, RD23, and TZZL1 are highlighted. Sequences r.