SummaryA total of 2 542 lincRNAs were identified from Populus trichocarpa and some of them play key roles in drought stress tolerance or regulate microRNA through target mimicry patterns.

BackgroundDNA methylation is an important biological form of epigenetic modification, playing key roles in plant development and environmental responses.ResultsIn this study, we examined single-base resolution methylomes of Populus under control and drought stress conditions using high-throughput bisulfite sequencing for the first time. Our data showed methylation levels of methylated cytosines, upstream 2kp, downstream 2kb, and repeatitive sequences significantly increased after drought treatment in Populus. Interestingly, methylation in 100 bp upstream of the transcriptional start site (TSS) repressed gene expression, while methylations in 100-2000bp upstream of TSS and within the gene body were positively associated with gene expression. Integrated with the transcriptomic data, we found that all cis-splicing genes were non-methylated, suggesting that DNA methylation may not associate with cis-splicing. However, our results showed that 80% of trans-splicing genes were methylated. Moreover, we found 1156 transcription factors (TFs) with reduced methylation and expression levels and 690 TFs with increased methylation and expression levels after drought treatment. These TFs may play important roles in Populus drought stress responses through the changes of DNA methylation.ConclusionsThese findings may provide valuable new insight into our understanding of the interaction between gene expression and methylation of drought responses in Populus.

BackgroundMicroRNAs (miRNAs) are endogenous small RNAs (sRNAs) with a wide range of regulatory functions in plant development and stress responses. Although miRNAs associated with plant drought stress tolerance have been studied, the use of high-throughput sequencing can provide a much deeper understanding of miRNAs. Drought is a common stress that limits the growth of plants. To obtain more insight into the role of miRNAs in drought stress, Illumina sequencing of Populus trichocarpa sRNAs was implemented.ResultsTwo sRNA libraries were constructed by sequencing data of control and drought stress treatments of poplar leaves. In total, 207 P. trichocarpa conserved miRNAs were detected from the two sRNA libraries. In addition, 274 potential candidate miRNAs were found; among them, 65 candidates with star sequences were chosen as novel miRNAs. The expression of nine conserved miRNA and three novel miRNAs showed notable changes in response to drought stress. This was also confirmed by quantitative real time polymerase chain reaction experiments. To confirm the targets of miRNAs experimentally, two degradome libraries from the two treatments were constructed. According to degradome sequencing results, 53 and 19 genes were identified as targets of conserved and new miRNAs, respectively. Functional analysis of these miRNA targets indicated that they are involved in important activities such as the regulation of transcription factors, the stress response, and lipid metabolism.ConclusionsWe discovered five upregulated miRNAs and seven downregulated miRNAs in response to drought stress. A total of 72 related target genes were detected by degradome sequencing. These findings reveal important information about the regulation mechanism of miRNAs in P. trichocarpa and promote the understanding of miRNA functions during the drought response.

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Phased, secondary, small interfering RNA (phasiRNA) perform essential biological functions in plants. However, limited information is available on the role of phasiRNA genes in Populus (poplar), especially during drought stress. In this study, we identified 20 PHAS loci generating 91 phasiRNA in the genome of the model forest tree Populus trichocarpa (P. trichocarpa; western balsam-poplar) using the control and drought libraries. Our analysis indicated that six PHAS (PtPHA14-20) initiated by two Populus-specific miRNAs (miR6445 and miR6427) were specific to Populus. In addition, a total of 47 phasiRNA were found to be drought responsive, and five of them were confirmed by RT-qPCR. The phase cleavage of three PHAS loci by miRNA, and degradation of nine transcript targets by phasiRNA were experimentally confirmed based on degradome data. The identification of these Populus phasiRNA will contribute to a better understanding of their function and regulation during drought stress.