Enhanced Cold Tolerance and Tillering in Switchgrass (Panicum virgatum L.) by Heterologous Expression of Osa-miR393a

The microRNA393 (miR393) family is one of the conserved miRNA families in the plant kingdom. MiR393 was reported to regulate rice tillering and abiotic stress resistance positively through an auxin signaling pathway. However, little is known about the function of miR393 in switchgrass (Panicum virgatum L.), an important bioenergy C-4 grass plant. We tested the expression level of miR393 and its four putative target genes (PvAFB1, PvAFB2, PvAFB3 and PvTIR1) in switchgrass, and found that these genes all responded to cold stress and exogenous 1-naphthaleneacetic acid (NAA) treatment. To investigate the function of miR393 in switchgrass, we enhanced miR393 expression by introducing an Osa-miR393a gene into switchgrass. The results showed that cold tolerance of the transgenic T-0 and T-1 generation plants was highly improved. Cold tolerancerelated genes PvCOR47, PvICE1 and PvRAV1 were negatively regulated by exogenous NAA, and the expression of these genes was significantly higher in transgenic plants than in wild-type plants. The transgenic T1 seedlings were more tolerant to exogenous NAA treatment, accumulating less H2O2 after cold treatments. It was also observed that the miR393/target module regulates cold tolerance responses in Arabidopsis. In addition, transgenic plants overexpressing miR393 had significantly more tillers and higher biomass yield per plant in greenhouse and field tests. Forage quality analyses revealed that the soluble sugar contents of transgenic plants were increased markedly. Overall, the results suggested that overexpression of miR393 improved cold tolerance and tillering of switchgrass through regulation of auxin signaling transduction.