Expressing a Short Tandem Target Mimic (STTM) of miR164b/e-3p enhances poplar leaf serration by co-regulating the miR164–NAC module

Abstract

MicroRNAs (miRNAs) are short non-coding RNAs (21–24 nt) that play important roles in plant growth and development. The miR164 family is highly conserved in plants and the miR164–NAM/ATAF/CUC (NAC) module is validated to regulate leaf and flower development, lateral root initiation and stress response. However, our knowledge of its role in Populus remains limited. In this study, two mature miRNA species, miR164e-5p and miR164e-3p, were identified in Populus deltoides. Their nucleotide sequences were identical to those of miR164a/b/c/d/e-5p and miR164b/e-3p in P. tremula × P. alba clone 717-1B4 (hereinafter poplar 717), respectively. Transgenic plants of poplar 717, including overexpression lines (35S::pri-miR164e) and Short Tandem Target Mimic lines (STTM-miR164a-d,e-5p and STTM-miR164b/e-3p), were generated to study the roles of miR164e-5p and miR164e-3p in poplar. Compared with poplar 717, the leaf margins of 35S::pri-miR164e lines were smoother, the leaves of STTM-miR164b/e-3p line were more serrated, while the leaf morphology of STTM-miR164a-d,e-5p lines had no obvious change. In addition, both 35S::pri-miR164e and STTM-miR164b/e-3p plants had a dwarf phenotype. Expressions of miR164a-d,e-5p target genes, including PtaCUC2a, PtaCUC2b and PtaORE1, was significantly reduced in the apex of 35S::pri-miR164e lines. Green fluorescent protein (GFP) reporter assay showed that PtaCUC2a/2b and PtaORE1 were cleaved by miR164a-d,e-5p, and the cleavage was inhibited by STTM-miR164b/e-3p. Therefore, miR164b/e-3p may cooperate with miR164a-d,e-5p to regulate certain NAC members, such as PtaCUC2a/2b and PtaORE1, thereby regulating leaf development and plant growth in poplar. Our findings add new insights into the mechanisms by which the miR164–NAC module regulates plant development.