Identification of microRNAs and their targets from Populus euphratica

Abstract

Populus euphratica is an ideal model system for research into the abiotic stress resistance research of woody plants. Although microRNAs have been found in poplars and have been shown to have diverse biological functions, a majority of them are genus- or specie-specific and few microRNAs have been found in P. euphratica to date. In this study, microRNA cloning and computational expressed sequence tag analysis were used to identify 72 putative miRNA sequences in P. euphratica. These sequences could be classified into 21 families, 12 of which were novel, increasing the number of known poplar microRNA families from 42 to 54. Expression analysis indicated that five P. euphratica microRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes are involved in development, resistance to stress, and other cellular processes. These results suggest several roles for miRNAs in the regulatory networks associated with the abiotic stress resistance of tree species.

Introduction

With sequencing of the Populus trichocarpa genome completed, the relatively small genome of poplar species and the large number of expressed sequence tags (EST) (∼428,901) available have facilitated genetic studies of trees. Poplar species are also easily transformed, regenerated, and propagated vegetatively [1]. Although the draft of the genome of P. trichocarpa is largely complete, the specific P. trichocarpa tree, from which the genome sequence was obtained, is no longer alive. However, as there are many other Populus species (∼30), suggesting the existence of genome diversity, research on Populus species besides P. trichocarpa has become of interest. The poplar species Populus euphratica is the only arboreal species that can become established in the world’s largest shifting-sand desert, the Taklimakan Desert [2], [3], which is characterized by wide temperature differences as well as salty, arid, and drought stress conditions [4], [5]. Thus, P. euphratica is widely considered as an ideal model system for researching into the abiotic stress resistance of woody plants [6]. Studies on this tree species will further our understanding of the resistance mechanism of woody plants to abiotic stress [7], [8], [9].

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, typically about 21 nucleotides in length, that repress gene translation or degrade target mRNAs and therefore have negative regulatory functions at the post-transcriptional level [10], [11]. A great deal of effort has gone into the identification of miRNAs in two model plants, Arabidopsis and rice. In the recently published miRBase 13.0, 112 and 143 miRNA families were described in Arabidopsis thaliana and Oryza sativa, respectively, whereas only 42 such families have been found in P. trichocarpa. Furthermore, most poplar miRNAs in the miRBase have been identified by comparing obtained sequences with genus-conserved miRNAs in A. thaliana and other genera, even though the majority of poplar miRNAs are genus- or species-specific [12]. Consequently, identifying novel and functional miRNA families in Populus have become of interest.

To date, the 234 poplar miRNAs registered in the miRBase 13.0 were obtained from data of five published papers [1], [13], [14], [15], [16]. Two of those studies found that the expression of a set of miRNAs is regulated by various abiotic stresses; thus, for example, in cold-stressed P. trichocarpa, Ptc-miR156g-j, Ptc-miR475a,b, and Ptc-miR476a were down-regulated while Ptc-miR168a,b and Ptc-miR477a,b were up-regulated [14]. In addition, ptr-miR156, 162, 164, 475, 480, 481, 408,159, 476, 479, 160, 172, and 168 were found to be differentially expressed in P. trichocarpa subjected to mechanical stress [14]. Although these miRNAs are associated with stress responses, few of this type have been identified. Furthermore, miRNA functional analyses have not been carried out in P. euphratica. Clearly, the identification of new miRNAs and of their functions in this species would help to elucidate their abiotic stress resistance mechanisms.

In this study, miRNAs in P. euphratica were identified in three ways. In the first, a small RNA cDNA library was constructed and sequenced, which led to the identification of miRNAs in the acquired small RNA sequences. In the second, poly A tails were added to the small RNAs, which were then reverse-transcribed in order to clone conserved miRNAs. Finally, the third method consisted of finding miRNAs in an expressed sequence tag (EST) database using a comparative genomic approach and sequence survey analysis based on the secondary structure of pre-miRNAs. A total of 72 putative miRNA sequences that could be classified into 21 families were identified from P. euphratica. Among them, 12 families were found to be novel and specific for P. euphratica. Expression analysis indicated that five P. euphratica miRNAs were induced by dehydration stress. Bioinformatics prediction showed that the 130 target genes were involved in development, resistance to stress, and other cellular processes.