Small RNA Differential Expression Analysis Reveals miRNAs Involved in Dormancy Progression in Sweet Cherry Floral Buds

Abstract

In sweet cherry (Prunus avium), as in other temperate woody perennials, bud dormancy allows for survival in adverse environmental conditions during winter. During this process, environmental signals such as short days and/or low temperatures trigger internal signals that enable buds to become tolerant to the cold. The process involves tracking chilling units up to chilling the requirement fulfillment to resume growth, a transition involving transcriptional regulation, metabolic signaling, and epigenetic-related regulatory events. Massive sequencing of small RNAs was performed to identify miRNAs involved in sweet cherry dormancy by comparing their expression in field (regular seasonal) and controlled non-stop (continuous) chilling conditions. miRNAs highlighted by sequencing were validated using specific stem-loop PCR quantification, confirming expression patterns for known miRNAs such as miR156e, miR166c, miR172d, miR391, miR482c, and miR535b, as well as for newly proposed miRNAs. In silico prediction of the target genes was used to construct miRNA/target gene nodes. In particular, the involvement of the sweet cherry version for the miR156/SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN genes whose expression was opposite in the two conditions suggests their involvement on dormancy regulation in sweet cherry. miRNA levels indicate that the regulation of stress-related genes and hormone synthesis modulates the expression of calcium metabolism and cell development-associated genes. Understanding the regulatory networks involved in sweet cherry dormancy, particularly in the context of miRNA involvement, represents the first step in the development of new agricultural strategies that may help overcome the increasing challenges presented by global climate change.