Salt‐stress priming enhances the tolerance of plants against subsequent exposure to a similar stress. Priming‐induced transcriptomic reprogramming is mediated by multiple epigenetic mechanisms, the best known of which is histone modifications. However, not much is known about other epigenetic responses. In this study, salt‐stress priming resulted in global DNA hypomethylation in the leaves of soybean seedlings. The DNA methyltransferase activities in primed seedlings were reduced, contributing to the overall DNA hypomethylation. Genes associated with the hypomethylated DNA regions in primed seedlings also showed a higher mean level of the active histone mark, histone 3 lysine 4 trimethylation (H3K4me3), and a lower mean level of the repressive histone mark, H3K4me2. Transcriptomic analyses supported that DNA hypomethylation played a role in fine‐tuning the chromatin status in primed seedlings to potentiate gene expressions. Motif and transcriptional network analyses revealed that DNA hypomethylation may facilitate the responses mediated by key transcription factors in the abscisic acid (ABA)‐dependent pathway. A pre‐treatment using a DNA methyltransferase inhibitor, 5‐azacytidine, could enhance salt tolerance in non‐primed soybean seedlings, similar to the priming effect, suggesting the role of DNA hypomethylation in salt‐stress priming. Overall, this research furthers our understanding of the epigenetic mechanisms involved in salt‐stress priming in soybean.

Summary: MicroRNAs (miRNAs) are important regulators of plant biological processes, including soybean nodulation. One miRNA, miR4407, was identified in soybean roots and nodules. However, the function of miR4407 in soybean is still unknown. MiR4407, unique to soybean, positively regulates lateral root emergence and root structures and represses a root‐specific ISOPENTENYLTRANSFERASE (GmIPT3). By altering the expression of miR4407 and GmIPT3, we investigated the role of miR4407 in lateral root and nodule development. Both miR4407 and GmIPT3 are expressed in the inner root cortex and nodule primordia. Upon rhizobial inoculation, miR4407 was downregulated while GmIPT3 was upregulated. Overexpressing miR4407 reduced the number of nodules in transgenic soybean hairy roots while overexpressing the wild‐type GmIPT3 or a miR4407‐resistant GmIPT3 mutant (mGmIPT3) significantly increased the nodule number. The mechanism of miR4407 and GmIPT3 functions was also linked to autoregulation of nodulation (AON), where miR4407 overexpression repressed miR172c and activated its target, GmNNC1, turning on AON. Exogenous CK mimicked the effects of GmIPT3 overexpression on miR172c, supporting the notion that GmIPT3 regulates nodulation by enhancing root‐derived CK. Overall, our data revealed a new miRNA‐mediated regulatory mechanism of nodulation in soybean. MiR4407 showed a dual role in lateral root and nodule development.