Network of miR396-mRNA in Tissue Differentiation in Moso Bamboo (Phyllostachys edulis)

MiR396 plays an essential role in various developmental processes. However, the miR396-mRNA molecular network in bamboo vascular tissue differentiation during primary thickening has not been elucidated. Here, we revealed that three of the five members from the miR396 family were overexpressed in the underground thickening shoots collected from Moso bamboo. Furthermore, the predicted target genes were up/down-regulated in the early (S2), middle (S3) and late (S4) developmental samples. Mechanistically, we found that several of the genes encoding protein kinases (PKs), growth-regulating factors (GRF), transcription factors (TFs), and transcription regulators (TRs) were the potential targets of miR396 members. Moreover, we identified QLQ (Gln, Leu, Gln) and WRC (Trp, Arg, Cys) d omains in five PeGRF homologs and a Lipase_3 domain and a K_trans domain in another two potential targets, where the cleavage targets were identified via degradome sequencing (p < 0.05). The sequence alignment indicated many mutations in the precursor sequence of miR396d between Moso bamboo and rice. Our dual-luciferase assay revealed that ped-miR396d-5p binds to a PeGRF6 homolog. Thus, the miR396-GRF module was associated with Moso bamboo shoot development. Fluorescence in situ hybridization localized miR396 in the vascular tissues of the leaves, stems, and roots of pot Moso bamboo seedlings at the age of two months. Collectively, these experiments revealed that miR396 functions as a regulator of vascular tissue differentiation in Moso bamboo. Additionally, we propose that miR396 members are targets for bamboo improvement and breeding.