Characterization and functional analysis of miR166f in drought stress tolerance in mulberry (Morus multicaulis)

MicroRNAs (miRNAs) regulate gene expression accurately and effectively at the posttranscriptional level by repressing translation or directly degrading target mRNAs. Over 40 plant miRNA family genes have been associated with abiotic stress; however, few reports have identified miRNAs and their molecular functions under abiotic stress in woody plants. Mulberry trees are ecologically and economically important perennial woody plants, but few studies have examined the stress physiology, biochemistry, and molecular biology of mulberry miRNAs. Previously, we identified miR166f in Morus alba and found that its target genes encode homeobox-leucine zipper (HD-Zip) transcription factors and histone arginine demethylase, which are involved in responses to heat, cold, drought, and salt stresses. In this study, we further characterized miR166f from mulberry, and found that it targets HD-Zip induced by drought stress. We isolated, cloned, and identified pre-miR166f, the precursor of miR166f, from the leaves of mulberry (Morus multicaulis); pre-miR166f was 91 bp in length with a minimum folding free energy of − 51.70 kcal/mol. Bioinformatics analysis showed that the 3000-bp 5′ upstream region of miR166f contained not only TATA and CAAT boxes that functioned in transcription initiation, but also had many cis-acting elements for stress responses, such as MBS, HSE, ARE, TC-rich repeats, and several hormones response elements, such as AuxRR-core, CGTCA-motif, and P-box. This analysis suggested that miR166f might participate in abiotic and biotic stress responses by regulating the expression of stress-related genes in mulberry. We constructed a miR166f binary overexpression vector, pCAMBIA-35S-GUS-miR166f, and established a transient transformation system in mulberry. Histochemical β-glucuronidase (GUS) staining of miR166f-overexpression in transient transgenic mulberry leaves showed the best effect and highest GUS gene expression compared to the wild-type control plants at day four posttransformation using an optimized concentration of transformation liquid (OD₆₀₀ = 0.7). The target genes of miR166f—two HD-Zips and one histone arginine demethylase gene (JMJD6)—showed markedly lower expression levels in miR166f-overexpression transient transgenic mulberry leaves. Further investigation showed that transient transgenic mulberry trees had higher relative water content, free proline content, soluble protein content, and superoxide dismutase and peroxidase activities, and lower malondialdehyde content compared to the wild-type control plants, which suggested that overexpression of miR166f in mulberry could enhance tolerance to drought stresses in transient transgenic mulberry. The results suggested that miR166f might function as a positive regulator of drought stress tolerance in mulberry.