Figure 8.

The regulation of gene expression by LjNLP4 and LjNIN. A, The effect of LjNLP4 or LjNIN overexpression on LjNF-YB expression. Real-time RT-PCR analysis of LjNF-YB transcripts in transgenic hairy roots produced from WT containing the LjUBQ_pro:GUS, LjUBQ_pro:LjNLP4, or LjUBQ_pro:LjNIN constructs. Transgenic roots were identified by GFP fluorescence. Each cDNA sample was prepared from total RNA derived from noninoculated roots incubated with 0 or 10 mM KNO₃ for 24 h (n = 3 independent pools of roots derived from 10 plants). The expression of LjUBQ was used as the reference. B–D, Transactivation of (B and C) 4xYB1:GUS and (D) LjNIR1_pro:GUS in L. japonicus mesophyll protoplasts transformed with respective constructs (n = 3 independent pools of protoplasts). B and C, Full length LjNLP4 (1‒976) and truncated LjNLP4 (LjNLP4ΔPB1; 1‒656) were expressed in the assay. Transformed protoplasts were incubated with (B and D) 0 or 10 or (C) 10 mM KNO₃. GUS activity was measured relative to (B and D) LjUBQ_pro:LUC or (C) 35S_pro:LUC activity. E, A schematic diagram of the promoter-GUS constructs used in (F) and (G). Promoter fragments containing the LjNLP4/LjNIN-binding site (blue bar) in the LjNIR1 promoter region were inserted upstream of the GUS gene. In the modified LjNIR1 promoter, LjNIR1(NF-YB)_pro, the LjNLP4/LjNIN-binding site was replaced with the LjNIN-binding site (red bar) from the LjNF-YB promoter region. F, Transactivation of LjNIR1_pro:GUS or LjNIR1(NF-YB)_pro:GUS in L. japonicus mesophyll protoplasts transformed with respective constructs (n = 3 independent pools of protoplasts). Transformed protoplasts were incubated with 10 mM KNO₃. G, Real-time RT-PCR analysis of GUS expression in WT transgenic hairy roots expressing each GUS construct. Transgenic roots were identified by GFP fluorescence. Each cDNA sample was prepared from total RNA derived from noninoculated roots incubated with 0 or 10 mM KNO₃ for 24 h (n = 3 independent pools of roots derived from five plants). The expression of GFP was used as the reference. Data of transactivation are normalized using the conditions in which GFP is expressed with (C and F) or without (B and D) nitrate. Individual data points are represented by blue points, and red crosses indicate the sample means. ^*P < 0.05 by a two-sided Student’s t test (A and B) or Welch's t test (D and G). Significant differences were determined with Kruskal–Wallis one-way analysis with Tukey multiple comparisons, indicated by different lower-case letters (P < 0.05; C and F).