A genome-wide identification of miPEPs in hybrid poplar reveals regulatory functions of miPEP166i in adventitious root elongation

MicroRNA-encoded peptides (miPEPs) activate their cognate miRNAs and regulate the growth and development of plants. They can also be chemically synthesized and applied as eco-friendly and safe growth regulators to improve agronomic traits. To explore the diversity and potential applications of miPEPs in forest trees, integrated peptidomics and genomics methods were applied to identify miPEPs in hybrid poplar 84 K (P. alba × P. tremula var. glandulosa). More than 1200 unique miPEPs from 544 pri-miRNAs were detected in leaf, stem, and root samples, covering 86% of the miRNA loci. Majority of the identified miPEPs were encoded by ORFs with non-AUG start codons. Among these miPEPs, about 30% located at less than 100 bp from the predicted transcription start sites (TSSs) of the miRNA loci. To characterize physiological functions of miPEPs in root development, miPEP166i, a root-specific miPEP, was synthesized and applied to growth media for poplar root regeneration. The result indicated that miPEP166i promoted the numbers and lengths of adventitious roots in comparison to peptides with random sequences. The expression of miR166i was induced by miPEP166i, which subsequently inhibited the levels of target genes in HD-ZIP III family. Transcriptional profiling data also indicated that genes in auxin synthesis and transport were induced by miPEP166i application, whereas genes encoding cytokinin-activating enzymes were inhibited. Our results provide a valuable resource for functional genomics research and perturbation of growth traits in woody plants.