Fig. 3: LjNIN regulates LjIMA1 expression. | Nature Communications

Fig. 3: LjNIN regulates LjIMA1 expression.

From: IMA peptides regulate root nodulation and nitrogen homeostasis by providing iron according to internal nitrogen status

Fig. 3

a Electrophoresis mobility shift assay (EMSA) showing LjNIN, LjNLP1, or LjNLP4 binding to the NIN-binding sequence (NBS)/nitrate-responsive cis-element (NRE), mutated NBS/NRE (mNBS/NRE) or NRE on the LjIMA1 or LjNIR1 promoter. Nucleotide sequences forming semi-palindromic structures are shown in red. MBP-LjNIN (551–878), MBP-LjNLP1 (573–903), or MBP-LjNLP4 (564–976) recombinant proteins, consisting of an RWP-RK and a PB1 domain, were incubated with the FAM-labeled DNA probe (Supplementary Data 3). NRE on the LjNIR1 promoter was used as a positive control. Three independent experiments were conducted to obtain similar results. b Transactivation of 4xNBS/NRE-35Smin:NanoLUC and 4xmNBS/NRE-35Smin:NanoLUC in L. japonicus mesophyll protoplasts (n = 4 independent pools of protoplasts). NanoLUC activity was measured relative to 35Spro:GUS activity. Transactivation data were normalized to the condition in which GFP was expressed. c, d RT-qPCR analysis of LjIMA1, LjIMA2, and LjNIN expression in the transgenic hairy roots carrying the construct that constitutively expressed LjNIN in the absence of rhizobia. Transgenic roots were identified by GFP signals and collected (n = 5 independent pools of hairy roots), and total RNA was isolated for cDNA synthesis. RT-qPCR data were normalized to the data of roots where an empty vector (EV) was introduced. e RT-qPCR analysis of LjIMA1/2 expression in the WT and Ljnin mutants. Non-inoculated or 3 dai shoots or roots were collected (n = 6 independent pools of shoots or roots derived from three plants). RT-qPCR data were normalized to WT non-inoculated conditions. The expression of LjUBQ was used as the reference. 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 c-e., bars indicate mean values. In b, e, different letters indicate statistically significant differences (P < 0.05, one- or two-way ANOVA followed by multiple comparisons). In c, d, asterisks indicate a statistically significant difference (P < 0.01, by a two-sided Mann–Whitney test). ns means not significant.

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