Mdm-miR160 Participates in Auxin-Induced Adventitious Root formation of apple rootstock

Adventitious root (AR) formation is an essential process for the vegetative propagation of apple rootstocks. However, the mechanisms of miR160 mediate AR formation in apple remains to be seen. In this study, five mdm-miR160 family members were identified in apple rootstocks and two AUXIN RESPONSIVE FACTOR (MdARF16 and MdARF17) were found as the targets of mdm-miR160. Furthermore, the sequences of mdm-miR160 family members' precursors were cloned from four different AR roofing ability apple rootstocks, whereas, mdm-miR160a, mdm-miR160c and mdm-miR160e were highly conserved than mdm-miR160b and mdm-miR160d. Mdm-miR160 and their targets expression patterns showed obviously negative relationship in different stages during AR formation. In addition, the transcript level of mdm-miR160 was higher in 'SH6' and lower in 'MP', where it responds to exogenous indole-3-butyric acid (IBA). Mdm-miR160a was cloned from 'M26' and used for further experiments. The result of over-expressed mdm-miR160a in tobacco (Nicotiana tabacum L.cv.SR1) inhibited AR formation in 35S:MdMIR160a transgenic lines as compared with WT, whereas, exogenous IBA treatment could promote AR formation in 35S:MdMIR160a transgenic lines. Expression of five endogenous miRNAs: miR167c, miR393a, miR396a, miR396b, and miR156 in transgenic lines were lower than in WT. Moreover, the targets of endogenous miRNAs in transgenic lines showed different expression patterns with WT. In consistent, mdm-miR160 was an auxin responsive factor and participated in AR formation in apple rootstock, however, over-expressed mdm-miR160a has inhibitory effect on AR formation while exogenous IBA treatment could raise the roofing ability of transgenic lines.