Fig. 4: N-dependent flowering regulation by Hd6-Hd2 module.

a Comparison of the DTH between NIL and the transgenic plants. n = 3, P-values were calculated by one-tailed paired t-tests. The box plot boundaries reflect the interquartile range; the centre line is the median and the whiskers represent 1.5× the interquartile range from the lower and upper quartiles. b Venn diagram of the genes downregulated by high N; adjusted P value < 0.05, logFC ≥ −1. c Visualization of GO enrichment analysis using 103 DEGs (low N > high N only in 35S:Hd6/Ubq:Ω:3FLAG-Hd2). All detected 20 GO terms are shown. d mRNA levels of Ehd1, Hd3a and RFT1 in NIL (hd6, hd16) and transgenic plants grown under different N conditions. Total RNAs were extracted from 55-day-old plants of T2 generation. Mean values with the SD. n ≥ 5 independent experiments. **P value < 0.01, *P value < 0.05 and n.s indicates no significant difference based on two-sided student’s t-test. e Effects of kinase inhibitors on 3FLAG-Hd2 accumulation. 35S:Hd6/Ubq:Ω:3FLAG-Hd2 seedlings were treated with different types of casein kinase inhibitor as the indicated concentration for 6 h. Each experiment was repeated independently for at least 3 times with similar results. f CIP treatment to crude extracts prepared from 35S:Hd6/Ubq:Ω:3FLAG-Hd2 seedlings. Crude extracts were applied to SDS-PAGE after incubation with or without CIP. Each experiment was repeated independently for at least 3 times with similar results. g Profile of 3FLAG-Hd2 protein in immunoblotting using SDS-PAGE containing phos-tag. Asterisk with the bracket indicates a smearing pattern of the 3FLAG-Hd2 signal under high N sample. 60 μg of crude extracts were loaded per lane for immunoblotting, reacted with anti-FLAG antibody. Each experiment was repeated independently for at least 3 times with similar results. Source data are provided as a Source Data file.