miR160 Interacts With Target Site and Negatively Regulates Its Expression During Conifer Somatic Embryo Development.

MicroRNAs (miRNAs) are key regulators of several plant developmental processes including embryogenesis. Most miRNA families are conserved across major groups of plant species, but their regulatory roles have been studied mainly in model species like and other angiosperms. In gymnosperms, miRNA-dependent regulation has been less studied since functional approaches in these species are often difficult to establish. Given the fundamental roles of auxin signaling in somatic embryogenesis (SE) induction and embryo development, we investigated a previously predicted interaction between miR160 and a putative target encoding in () embryonic tissues. Phylogenetic analysis of () from and , used here as a model system of conifer embryogenesis, showed their close relatedness to () genes known to be targeted by miR160 in other species, including Arabidopsis and . By using a luciferase (LUC) reporter system for miRNA activity in protoplasts, we have confirmed that miR160 (ppi-miR160) interacts with target site. When the primary miR160 from was overexpressed in protoplasts under non-limiting levels of ARGONAUTE1, a significant increase of miR160 target cleavage activity was observed. In contrast, co-expression of the primary miRNA and the target mimic led to a decrease of miR160 activity. Our results further support that this interaction is functional during consecutive stages of SE in the conifer model . Expression analyses conducted in five stages of development, from proembryogenic masses (PEMs) to the mature embryo, show that conifer is negatively regulated by miR160 toward the fully developed mature embryo when miR160 reached its highest expression level. This study reports the first validation of a predicted target site of a conifer miRNA supporting the conservation of miR160 interaction with targets in gymnosperms. The approach used here should be useful for future characterization of miRNA functions in conifer embryogenesis.