Fig. 5: LjIMA1/2 is required for nitrogen homeostasis depending on the nitrogen–Fe balance.

a, b RT-qPCR analysis of LjIMA1/2 expression in response to nitrogen nutrients. Shoots were collected from WT plants treated with 5 mM KNO₃, 5 mM NH₄Cl, or 2.5 mM NH₄NO₃ for 2 d in the absence of rhizobia (n = 3 independent pools of shoot from three plants). RT-qPCR data were normalized to non-treated conditions. The expression of LjUBQ was used as the reference. c–f Shoot phenotypes, shoot FW (n = 11 or 12 plants) and shoot nitrate (n = 4 independent pools of shoot derived from 2 or 3 plants) and shoot chlorophyll contents (n = 4 independent pools of shoot derived from 2 or 3 plants) of WT and Ljima1/2 grown in different nitrogen–Fe ratios for 17 d after germination in the absence of rhizobia. KNO₃ and Fe (III)-EDTA were used for nitrogen and Fe sources, respectively. Scale bar, 1 cm. Centerlines in the boxplots show the medians, and box limits indicate the 25th and 75th percentile. The whiskers go down to the smallest value and up to the largest. Scatterplots show individual biological replicates as dots. In a, b, bars indicate mean values. In a, b, different letters indicate statistically significant differences (P < 0.05, one-way ANOVA followed by multiple comparisons). In d–f, different letters indicate statistically significant differences (P < 0.05, two-way ANOVA followed by multiple comparisons).