Deciphering the regulatory network of miR156 in plant architecture and abiotic stress resistance of alfalfa (Medicago sativa) by transcriptome sequencing

To improve alfalfa (Medicago sativa) biomass yield and abiotic stress resistance is a challenge due to its autotetraploidy and self-incompatibility. Previously we have generated transgenic alfalfa lines overexpressing miR156 and showing excellent plant architecture. In this study, we investigated the regulation network of miR156 in these transgenic alfalfa lines. Based on 83,202 non-redundant full-length transcripts in a pool of roots, stems, leaves, flowers, pods, and apical buds, we identified 154 candidate genes as potential downstream targets of miR156, especially for M. sativa SQUAMOSA promoter binding protein-like genes (MsSPLs), and then identified 26 MsSPL gene family members in alfalfa genome. The expressions of MsSPL5B and MsSPL3 were downregulated in root and leaf respectively, whereas that of MsSPL4 was significantly downregulated in stem, leaf, root, and apical bud tissues due to miR156 overexpression. We revealed the key role of MsSPL4-mediated regulatory pathways in the substantial correlations between agronomic traits and gene expression, and further found several involved hub genes showing significant correlation with MsSPL4. In addition, MsSPL4 overexpression in tobacco enhanced salinity tolerance but inhibited bud induction. Our results provide strong evidence for understanding the roles of miR156 and MsSPLs in regulating plant architecture and abiotic stress responses in alfalfa.