Fig. 4: LjIMA1/2-mediated Fe provision is required for root nodule symbiosis.

a Transcriptome analysis of Fe-related genes in response to LjIMA1/2 overexpression. Transcriptome data were obtained from the WT plants with transgenic hairy roots carrying control empty vector (EV), LjUBQ_pro:LjIMA1 or LjUBQ_pro:LjIMA2 constructs 5 dai (n = 3 independent pools of hairy roots from three plants). The expression patten of LjIRT1 (Lj4g3v1428550), LjVIT (Lj4g3v0336390), LjNAS2 (Lj3g3v1048990), LjFRO2a (Lj0g3v0105009), LjFRO2b (Lj0g3v0360999), LjFIT1a (Lj0g3v0107279), LjFIT1b (Lj3g3v1113460), LjIMA1 (Lj2g3v3071890), and LjIMA2 (Lj0g3v0238729) are shown. b Fe amounts of non-inoculated or 14 dai shoots or roots in WT and Ljima1/2 (n = 3 or 4 independent pools of shoots or roots derived from 12 plants). c. Perls Prussian Blue Fe staining of 14 dai nodules in WT and Ljima1/2. Three independent experiments were conducted to obtain similar results. d, f Nodulation phenotypes of WT and Ljima1/2 14 dai in different Fe concentrations. d Total nodule number (n = 12 plants). e Maximum nodule diameter of nodules on the root of each plant (n = 12 plants). g RT-qPCR analysis of LjNAS2 expression in WT and Ljima1/2. Non-inoculated (0 dai) or 5 dai roots were collected (n = 3 independent pools of roots derived from three plants). RT-qPCR data were normalized to WT 0 dai conditions. h Relative nicotianamine amount in WT and Ljima1/2 nodules 21 dai (n = 4 independent pools of roots derived from 12 plants). Data were normalized to nicotianamine amount in WT. i RT-qPCR analysis of LjIMA1/2 expression in WT. Shoots or roots of 11 dai plants with 1 mM Fe (+Fe) or without Fe (−Fe) were collected (n = 8 independent pools of shoots or roots derived from three plants). RT-qPCR data were normalized to –Fe conditions. The expression of LjUBQ was used as the reference. Fe (III)-EDTA was used for the Fe source. Scale bars, 500 µm in (c) and 2 mm in (f). 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 b, error bars indicate SEM. In g, i, bars indicate mean values. In b, d, e, different letters indicate statistically significant differences (P < 0.05, two-way ANOVA followed by multiple comparisons). In g, different letters indicate statistically significant differences (P < 0.01, one-way ANOVA followed by multiple comparisons). In h, i, asterisks indicate a statistically significant difference (P < 0.01, by a two-sided Welch’s t test or Mann–Whitney test).