Genome-Wide Identification of Copper Stress-Regulated and Novel MicroRNAs in Mulberry Leaf.

Copper (Cu) is an essential trace element for plant growth and development. It is widely involved in respiration, photosynthesis, pollen formation, and other biological processes. Therefore, low or excessive copper causes damage to plants. Mulberry is an essential perennial economic tree. At present, research on the abiotic stress responses in mulberry is mainly focused on the identification of resistant germplasm resources and cloning of resistant genes. In contrast, studies on the resistance function of microRNAs and the regulatory gene responses to stress are rare. In this study, small RNA libraries (control and copper stressed) were constructed from mulberry leaf RNA. High-throughput sequencing and screening were employed, a total of 65 known miRNAs and 78 predicted novel mature miRNAs were identified, among which 40 miRNAs were differentially expressed under copper stress. Subsequently, expression patterns were verified for 14 miRNAs by real-time fluorescence quantitative PCR (qPCR). The target genes of miRNAs were validated by 5' RLM-RACE. Our results provide the bases for further study on the molecular mechanism of copper stress regulation in mulberry.

Functional Characterization of MaZIP4, a Gene Regulating Copper Stress Tolerance in Mulberry (Morus atropurpurea R.).

ZIP4 (zinc transporter 4) plays important roles in transporting Cu2+ ions in plants, which may contribute to the maintenance of plant metal homeostasis in growth, plant development and normal physiological metabolism. However, ZIP4 transporters have not been described in mulberry and the exact function of ZIP4 transporters in regulating the homeostasis of Cu in mulberry remains unclear. In this study, a new ZIP4 gene (MaZIP4) was isolated and cloned from Morus atropurpurea R. Phylogenetic analysis of amino sequences suggested that the amino-acid sequence of the MaZIP4 protein shows high homology with other ZIP4 proteins of Morus notabilis, Trema orientale, Ziziphus jujube and Cannabis sativa. In addition, a MaZIP4 silenced line was successfully constructed using virus-induced gene silencing (VIGS). The analysis of MaZIP4 expression by quantitative real-time PCR in mulberry showed that the level of MaZIP4 expression increased with increasing Cu concentration until the Cu concentration reached 800 ppm. Relative to the blank (WT) and the negative controls, malondialdehyde (MDA) levels increased significantly and rose with increasing Cu concentration in the MaZIP4 silenced line, whereas the soluble protein and proline content, superoxide dismutase (SOD) and peroxidase (POD) activities of these transgenic plants were lower. These results indicated that MaZIP4 may play an important role in the resistance of mulberry to Cu stress.