Dormancy is a mechanism evolved in woody perennial plants to survive the winter freezing and dehydration stress via temporary suspension of growth. We have identified two aspen microRNAs (ptr-MIR169a and ptr-MIR169h) which were highly and specifically expressed in dormant floral and vegetative buds. ptr-MIR169a and its target gene PtrHAP2-5 showed inverse expression patterns during the dormancy period. ptr-MIR169a transcript steadily increased through the first half of the dormancy period and gradually declined with the approach of active growing season. PtrHAP2-5 abundance was higher in the beginning of the dormancy period but rapidly declined thereafter. The decline of PtrHAP2-5 correlated with the high levels of ptr-MIR169a accumulation, suggesting miR169-mediated attenuation of the target PtrHAP2-5 transcript. We experimentally verified the cleavage of PtrHAP2-5 at the predicted miR169a site at the time when PtrHAP2-5 transcript decline was observed. HAP2 is a subunit of a nuclear transcription factor Y (NF-Y) complex consisting of two other units, HAP3 and HAP5. Using digital expression profiling we show that poplar HAP2 and HAP5 are preferentially detected in dormant tissues. Our study shows that microRNAs play a significant and as of yet unknown and unstudied role in regulating the timing of bud dormancy in trees. (C) 2013 Elsevier Inc. All rights reserved.