Identification of drought-responsive and novel Populus trichocarpamicroRNAs by high-throughput sequencing and their targets using degradome analysis
© Shuai et al.; licensee BioMed Central Ltd. 2013
Received: 15 August 2012
Accepted: 27 March 2013
Published: 9 April 2013
MicroRNAs (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.
Two 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.
We 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.
KeywordsPopulus trichocarpa microRNA Drought Target identification
MicroRNAs (miRNAs) are one of the most abundant classes of small RNAs (sRNAs) in plants and animals. These endogenous sRNAs were first identified in a metazoan called Caenorhabditis elegans in 1994  and were subsequently identified in plants  and viruses . MiRNAs are typically 21 nucleotides (nt) in length and play regulatory roles at the post-transcriptional level by repressing translation or directly degrading target message RNAs (mRNAs) . Plant miRNA genes are first transcribed into primary miRNAs, and then processed into miRNA precursors with stem-loop structures by Dicer-like proteins. Finally, they are released into the cytoplasm by cleavage into an miRNA::miRNA* duplex from the nucleus . The mature miRNAs join an RNA-induced silencing complex (RISC), and the RISC targets specific mRNAs and downregulates the expression of target mRNAs . MiRNAs participate in various processes such as metabolism , growth , development [9, 10], biotic  and abiotic [12–19] stress tolerance.
An increasing body of evidence indicates that miRNAs are involved in the plant drought stress response [13–15, 17, 20, 21]. In Arabidopsis, four drought-responsive miRNAs (miR396, miR168, miR167, and miR171) have been identified by microarray analysis . In tobacco, nine miRNAs strongly induced by drought stress have been experimentally identified, among which miR395 and miR169 are the two miRNAs most sensitive to drought stress . In rice, 30 miRNAs have been identified as significantly down- or upregulated under drought stress using a microarray platform . In Medicago truncatula (M. truncatula), Wang et al. (2011) mined drought-responsive miRNAs on a genome-wide scale using the Illumina sequencing technology; 22 members from four miRNA families and 10 members of six miRNA families were identified as up- and downregulated in response to drought, respectively . Li et al. (2011) reported 104 upregulated and 27 downregulated miRNAs by Illumina sequencing and microarray profiling in Populus euphratica (P. euphratica) . Furthermore, Qin et al. (2011) confirmed three upregulated and two downregulated mature miRNAs in response to drought using a RT-qPCR assay .
Environmental stressors due to climate change, especially drought stress, could make forests increasingly vulnerable to disease and die-offs . Drought may have a profound effect on forest health . With its modest genome size and rapid, widespread growth, P. trichocarpa was the first model forest species sequenced . Lu et al. (2005) studied miRNAs in P. trichocarpa and identified stress-responsive and novel miRNAs by Sanger sequencing technology . An additional 15 novel P. trichocarpa miRNAs were further identified by Klevebring et al. (2009) using the 454 sequencing method . Further study is needed to elucidate the mechanism of regulation of P. trichocarpa miRNA in general and of drought-responsive miRNAs in particular.
Only 234 P. trichocarpa miRNA precursors are annotated in the miRBase (version 18.0) , compared to 581 and 635 for Oryza sativa and M. truncatula, respectively, two other model organisms. Since the genome size of P. trichocarpa (423 Mbp, JGI version 3.0) is similar to that of M. truncatula (approximately 454–526 Mbp) and rice (389 Mbp), the potential for identification of new, specific miRNAs in P. trichocarpa is great. In this context, high-throughput sequencing was used to identify non-conserved miRNAs and drought-responsive miRNAs with the new version of the poplar genome (version 2.0), which has not been used in previous research on P. trichocarpa. The targets of these conserved and novel miRNAs were predicted, and some of them were confirmed by degradome sequencing. We discussed the potential regulatory mechanism between miRNAs and their targets. This may help to unravel the mechanism of drought stress tolerance in P. trichocarpa and other plants.
Illumina sequencing of P. trichocarpaleaves under control and drought conditions
Sequencing of miRNAs in Populus plants
Redundant reads (x10000)
Unique reads (x10000)
leaf and stem
After genomic annotation of the P. trichocarpa sRNAs, small interfering RNA (siRNA) and miRNA with various important post-transcription regulating functions were the largest of our acquired sequences. The siRNA is a 22 to 24 nt double-strand RNA, each strand of which is 2 nt longer than the other on the 3’ end . These aligned sequences might represent siRNA candidates. In total, deep sequencing obtained 577,393 and 956,979 siRNA candidates after the control and drought stress treatments, respectively (Additional file 1: file S1). Interestingly, the ratio of siRNA reads to all sRNAs reads increased sharply from 2.20% (CL) to 3.17% (DL). To obtain the annotation of known miRNAs, sRNAs were aligned to the miRBase 18.0 of P. trichocarpa. In total, 10,784,410 and 15,674,365 sequencing reads were identified as known poplar miRNAs in the two libraries. Thirty-four families from 207 known miRNAs were found, which accounted for about 87.3% of the total members. The remaining 30 miRNAs were not detected (Additional file 2: S2), possibly because of the tissue specificity of expression in poplar.
Novel non-conserved miRNAs from P. trichocarpa
New miRNAs in P. trichocarpa
Differential expression of miRNAs in P. trichocarpa
We further analyzed the expressions of the 65 new miRNAs under the two treatments. The drought-responsive miRNAs are listed in Figure 3; all were confirmed by the sequencing and RT-qPCR results. Among the 65 miRNAs, two novel miRNAs (Ptc-miRn6a-d and Ptc-miRn16) were downregulated by drought stress, and only miRn5 was upregulated in response to drought stress (Additional file 5: S5).
Target analysis of novel and conserved miRNAs by degradome sequencing
The previously known miRNA targets also identified in this study are available on the PopGenIE site (http://bioinformatics.cau.edu.cn/PMRD/adjunct/ptc_miR_target.txt). For new miRNAs whose targets were not known, we predicted their targets using the plant target prediction pipeline by the P. trichocarpa genome V2.0. The rules used for target prediction were based on those suggested by Allen et al. (2005)  and Schwab et al. (2005), as follows: (i) no more than four mismatches between the sRNA and the target (G-U bases count as 0.5 mismatches); (ii) no more than two adjacent mismatches in the miRNA/target duplex; (iii) no adjacent mismatches in positions 2–12 of the miRNA/target duplex (5’ of miRNA); (iv) no mismatches in positions 10–11 of the miRNA/target duplex; (v) no more than 2.5 mismatches in positions 1–12 of the miRNA/target duplex (5’ of miRNA); and (vi) the minimum free energy (MFE) of the miRNA/target duplex should be equal or greater than 74% of the MFE of the miRNA bound to its perfect complement . We predicted 281 targets for 53 miRNA families; the other six were not found (Additional file 6: S6).
Targets of P. trichocarpa miRNAs verified by degradome sequencing
Methionine sulfoxide reductase B 1
PS II oxygen-evolving complex 1
PS II oxygen-evolving complex 1
NOP56 (Arabidopsis homolog of
Eukaryotic translation initiation factor 4F
Glycoprotease M22 family
Chlororespiration reduction 1
LOX2 (lipoxygenase 2)
LOX2 (lipoxygenase 2)
FAT domain-containing protein
LOX2 (lipoxygenase 2)
PETE1 (plastocyanin 1)
VEP1 (vein patterning 1)
Zinc finger (CCCH-type) family protein
Fluorescence increase protein
Translation initiation factor
ATP-dependent Clp protease
Carotenoid cleavage dioxygenase 1
Carotenoid cleavage dioxygenase 1
Photosystem I subunit L
Preprotein translocase secA
Unfertilized embryo sac 10
16S rRNA processing
UBQ10 (Polyubiquitin 10)
APT1 (Adenine phosphoribosyl
Abnormal inflorescence meristem
Heat shock protein 81-4
SKP2A (F-box protein)
Beta-catenin repeat family protein
PEX5 (Peroxin 5)
termination factor family protein)
Thylakoidal ascorbate peroxidase
Function category of the identified target transcripts
Number of targets
Regulation of transcription
High-throughput sequencing of Populus
In a comparison of six Populus miRNA studies (Table 1) [11, 15, 25, 26, 29, 30], two used traditional Sanger sequencing [25, 29], two others used 454-pyrosequencing [26, 30], and the remaining two used the latest Illumina sequencing technology (as in the present study) [11, 15]. Along with the rapid development of sequencing technology, CL and DL can result in more sequences and greater sequencing depths than those reported in previous publications, due to the high throughput of the Illumina sequencer. In our study, because of the in-depth search, a large number of novel non-conserved miRNAs were found. The P. trichocarpa genome of Version 2.0 was used in this study; the transcript assemblies of the P. trichocarpa genome Version 2.0 are more meticulous than those of Version 1.1. This can increase the likelihood of finding more new miRNAs in general and drought-induced novel miRNAs in particular.
Compared to six previous studies of Populus plants [10, 11, 15, 18, 19, 26], we identified 28 novel miRNAs have been identified (Table 2). Eleven of these were found at least once. On comparing the miRNA counts, 24 had counts greater than 100. Interestingly, two of the members of the Ptc-miRn54 family are the most frequently and robustly miRNAs identified in poplar high-throughput sequencing studies. Furthermore, the counterparts of Ptc-miRn40, Ptc-miRn52, Ptc-miRn54a, and Ptc-miRn54b in P. beijingensis were verified by RT-qPCR . This provides more, strong evidence for the novel miRNAs identified from P. trichocarpa.
Drought-responsive miRNAs in P. trichocarpa
MiRNAs responsive to drought stress in diverse plant species
Drought in other publication(↑&↓)
Arabidopsis thaliana(↑), Nicotiana tabacum(↓), Oryza sativa(↓), Panicum. Virgatum(↑)
Populus tomentosa(↑&↓), Saccharum spp. (↑)
Medicago truncatula(↓), S. spp. (↑&↓)
Glycine max(↑), P. tomentosa(↑), S. spp. (↓)
Populus euphratica(↓),P. tomentosa(↑)
A. thaliana(↑), Hordeum vulgare(↑), M. truncatula(↑), O. sativa(↓), P. tomentosa(↓)
We further studied the target genes of these drought-responsive miRNAs by sequencing of the degradome library and comparing our work to previous studies [25, 29]. We found two upregulated miRNAs (Ptc-miR472 and Ptc-miRn5) that were both predicted to target putative disease resistance proteins in P. trichocarpa (Additional file 5: S5) . The cross adaptation between disease resistance and drought stress tolerance in plants exists through unknown mechanisms. Ptc-miR159 is another upregulated miRNA; its Arabidopsis homolog targets an MYB transcription factor. The ABA-induced accumulation of the miR159 homolog makes the MYB transcript degradation desensitize hormone signaling during seedling stress responses in Arabidopsis . According to our degradome sequencing results, the Ptc-miR159 was confirmed to target a methionine sulfoxide reductase (MSR). The homologs of MSR were induced by biotic and abiotic stresses in plants [47–50]. They catalyze the reduction of methionine sulfoxide to methionine  and play a major role in regulating the accumulation of reactive oxygen species (ROS), which can damage proteins in plant cells . Regulation of the MSR gene by Ptc-miR159 may occur through a homeostatic mechanism in response to drought stress in P. trichocarpa.
Ptc-miR473 was also upregulated in drought stress. It targets a member of a plant-specific GRAS transcription factor gene family . Another member of this family (PeSCL7) from P. euphratica was confirmed to play key roles in salt and drought stress tolerance . In the present study, Ptc-miR473 was confirmed to be targeted to Vein Patterning 1 (VEP1), which belongs to a short-chain dehydrogenase/reductase (SDR) superfamily . The homolog of VEP1 in Arabidopsis was confirmed to be required for vascular strand development and to be upregulated by osmotic stress [52, 53]. Ptc-miR473 regulates the expression the GRAS protein and VEP1, both of which were responsive to drought stress, this may be the drought tolerance mechanism in P. trichocarpa.
The number of downregulated miRNAs was larger than the number of upregulated miRNAs. The two downregulated miRNAs (miR160 and miR164) were both identified to be cold-responsive miRNAs in P. trichocarpa . TMV-Cg virus infection in Arabidopsis causes the accumulation of miR160 and miR164 . Three auxin responsive factor (ARF) genes (ARF10, ARF16, and ARF17) are the targets of miR160 . Repression of ARF10 by miR160 is critical for the seed germination and post-germination stages . MiR164 has been predicted targete six NAC-domain proteins (PNAC041, PNAC042, PNAC151, PNAC152, PNAC154, and PNAC155) from subfamily NAC-a , and NAC-domain proteins have been confirmed to be important in drought stress tolerance [58, 59]. These mechanisms may also be at work in drought-stress tolerance in P. trichocarpa for these two miRNAs.
Two downregulated miRNAs (Ptc-miR408 and Ptc-miR1444) have been reported to be Cu-responsive miRNAs in P. trichocarpa. Their targets include miR408-targeted plastocyanin-like proteins and miR1444-targeted all plastid polyphenol oxidases [60, 61]. Drought treatment may increase the relative concentration of Cu ion in the cytoplasm. When the Cu supply is sufficient, it is envisaged that the conjunction between mature miRNAs and their precursors will be suppressed, leading to the upregulation of miRNA-targeted Cu proteins . Accordingly, the balance of Cu ion contributes to the healthy growth and development of poplars during stress. In P. trichocarpa, Ptc-miR1444a is reportedly downregulated by dehydration , and Ptc-1444b/c was also found to be downregulated by drought in this study. MiR408 is reportedly downregulated by drought stress in rice  and has been experimentally identified to target an early responsive dehydration-related (ERD) protein in P. trichocarpa. Drought stress might induce the expression of ERD protein by downregulating the expression of miR408 in P. trichocarpa. This may be one of the mechanisms of regulation of drought-stress tolerance .
Other downregulated miRNA is Ptc-miR394, whose predicted targets are annotated as dehydration-responsive protein (POPTR_0002s07760.1) and F-box proteins (POPTR_0001s13770.1 and POPTR_0003s16980.1), which were recently reported to be differentially regulated by stress conditions and to play significant roles in the abiotic stress-response pathway. In Arabidopsis, salt-induced miR394 targets the mRNA of F-box proteins [12, 56].
From the analysis of predicted targets to downregulated Ptc-miRn6, a CCCH-type zinc finger protein and two trichome birefringence-like proteins (TBLPs) were functionally predicted. Although a cotton CCCH-type zinc finger protein has been identified to enhance abiotic stress tolerance in tobacco , we did not find any additional possible regulatory mechanisms between CCCH-type zinc finger protein and drought tolerance in P. trichocarpa. The homolog of TBLP in Arabidopsis is important to the formation of crystalline cellulose in trichomes . As previous studies have reported, trichome density increases with water shortage , and the thick trichome layer could prevent water loss . This may be the mechanism by which miRn6 regulates the expression of TBLP to adapt to drought stress.
Degradome analysis of non-drought-responsive miRNAs
In Arabidopsis, miR390 was reported to target TAS genes , while in P. trichocarpa, no TAS homologs have been found . From our study, the degradome sequencing data proved the adjustment mechanism of Ptc-miR390 and lipoxygenases (LOXs). The activity of LOX protein can partially reduce the production of radicals and ROS . This may explain the regulatory mechanism of miR390 in poplars. Four UDPGs were found to be targeted by Ptc-miR482, and all were classified as category I. The UDP-glucosyltransferases (UDPGs) are enzymes that attach a sugar molecule to a specific acceptor in plants . As in Arabidopsis, the UDPG is a key regulator of stress adaption through auxin IBA  and plays a role in fine-tuning nitrogen assimilation in cassava . This is a novel mechanism by which miR482 regulates the UDPG gene family in P. trichocarpa.
The degradome sequencing results imply that the miRNAs with no detected targets may silence genes by repressing translation. However, we could not obtain information about translation repression by miRNA through degradome sequencing. Only 19 targets of new miRNAs were identified. The targets of these non-conserved miRNAs are difficult to detect, possibly because of low abundance or a spatial expression pattern. More studies are needed to shed light on the regulation network of these miRNAs in P. trichocarpa. Over-expressing or repressing expression of these miRNAs in P. trichocarpa may help to elucidate the regulation mechanism.
In this study, sRNA libraries and degradome libraries of control and drought treatments were constructed with poplar leaves for high-throughput sequencing. Twelve miRNA members in 11 families were confirmed to be responsive to drought stress, and 65 novel miRNAs with star sequences of 59 families were identified. Through degradome sequencing, 53 and 19 genes were identified as cleavage targets of annotated miRNAs and new miRNAs, respectively. The functions of miRNA targets were analyzed and discussed. This study provides useful information for further analysis of plant miRNAs and drought stress tolerance, particularly in Populus plants.
Plant materials and total RNA extraction
P. trichocarpa seedlings of the same size (~5 cm) from tissue culture were planted in individual pots (15 L) containing loam soil and placed in a greenhouse at Beijing Forestry University. They were well irrigated and grown under control conditions (25°C day/20°C night, 16-h photoperiod) for three months, the heights of them were about 45 cm. During the period of drought-stress treatment, P. trichocarpa seedlings were sustained at two RSMC levels (70–75% and 15–20%) for 1 month according to a previous publication . The mature leaves were used as drought materials. Mature leaves from soil with sufficient irrigation (RSMC at 70–75%) were used as a control, and a relatively modest dehydration level (RSMC at 15–20%) was chosen for the drought treatment. Each treatment contained three repeat individuals. Leaf water potential (WP) was measured by PsyPro WP data logger (Wescor) (Additional file 8: S9). Photosynthetic rate, water conductance, intercellular CO2 concentration, and transpiration rate were measured by Li-6400 Photosynthesis System (Li-Cor) (Additional file 9: S10). For material harvest, mature leaves from the same position of different individual plants were collected and frozen immediately in liquid nitrogen for RNA extraction. The total RNA was extracted by the standard CTAB method for plants . Then they were used for sequencing and RT-qPCR.
High-throughput sequencing and bioinformatics analysis
Illumina sequencing on sRNAs (ranged from 18 nt to 30 nt) was conducted using an Illumina Genome Analyzer, following the Illumina protocol . After removing contaminants, low-quality sequences, and <18 nt sequences, clean reads were obtained and aligned against the P. trichocarpa genome (version 2.0) using SOAP software . tRNA, rRNA, snRNA, snoRNA, and some other repeat sequences were removed from the sequences with a perfect match to the genome through a search of the NCBI Genbank and Rfam databases . The remaining unique sequences were divided into known miRNAs and candidate miRNAs by alignment with the miRbase 18.0 . The candidate miRNAs were further analyzed by MFOLD software on the RNA secondary structure of the miRNA::miRNA* and pre-miRNA hairpin energy . Parameters were set to meet the criteria of plants .
Differentiatial expression analysis of miRNAs between the two treatments
The sequence reads of the two libraries were normalized to 1 million by the total number of sRNA reads in each sample. The calculation of the p-value for comparison of the miRNA expression between the two libraries was based on previously established methods [77, 78]. Specifically, the log2 ratio formula was: log2 ratio = log2 (miRNA reads in drought treatment/miRNA reads in control).
where N1 is the total number of reads in the sequencing library of the control, N2 is the total number of reads in the sequencing library of the drought treatment, x is the number of reads for an miRNA in the control library, and y is the number of reads for an miRNA in the drought treatment library.
All calculations were performed on a BGI Bio-Cloud Computing platform (http://www.genomics.cn/en/navigation/show_navigation?nid=4143). Normalized miRNAs of <1 were filtered in both libraries.
RT-qPCR of mature miRNAs
To validate the results of miRNAs from high-throughput sequencing, RT-qPCR was performed. The RNAs were extracted from leaves using the CTAB method . A poly (A) was added to the 3’ end, and reverse transcription was begun. In particular, a known sequence at the 5’ end of the oligo-dT primer was designed to be a communal reverse primer of the RT-qPCR. The One Step Prime-Script miRNA cDNA Synthesis Kit and SYBR Premix ExTag II (TaKaRa) were used. All primers used in this study are listed in Additional file 10: S8. The 5.8S ribosomal RNA was used as the internal control . RT-qPCR was performed using an ABI StepOnePlus instrument. Calculation of RT-qPCR results were revised as follow: Sample cycle threshold (Ct) values were determined and then standardized based on the 5.8S gene control primer reaction, and the 2-ΔΔCT method was applied to calculate the relative changes in gene expression from RT-qPCR experiments .
Target prediction and confirmation by degradome sequencing
New P. trichocarpa miRNA targets were predicted as described before [36, 80–82]. During the prediction, a penalty score (alignment score) criterion was induced according to the alignment between the miRNA and its potential target. Our cut-off values in both prediction and degradome sequencing data analysis were also set to <2.5 as used in previous studies on poplar miRNA target prediction. The biological function of the predicted targets was retrieved from the Universal Protein Resource (http://www.uniprot.org).
Degradome sequencing following the PARE protocol was used . Only miRNA-cleaved mRNA and other degraded mRNA could be ligated by a 5’ RNA adapter because the 5’-phosphate and intact mRNAs were protected by the 5’ cap. First, adapters and low-quality nucleotide reads were removed from raw reads using the Fastx-Toolkit. Then the clean reads were further analyzed by Cleaveland 2.0 software . Briefly, the reads were first mapped to the P. trichocarpa transcripts database from JGI Phytozome 2.0. At this step, a target plot was also created to distinguish the true miRNA cleavage site from background noise. We ran Cleaveland 2.0 with default parameters using 100 randomized sequencing shuffles. The NCBI database was used to predict functions of targets that were not annotated in JGI Phytozome 2.0. The cleaved target transcripts were categorized into three categories according to the following criteria: I, the abundance of reads in its cleavage site is the maximum on the transcript; II, the abundance of reads in its cleavage site is not the maximum, but is equal to or higher than the median for the transcript; and III, the abundance of reads in its cleavage site is less than the median for the transcript.
- P. trichocarpa:
RNA-induced silencing complex
- M. truncatula:
- P. euphratica:
Small interfering RNA
Minimum free energy
Quantitative real time polymerase chain reaction
- P. beijingensis:
Methionine sulfoxide reductase
Vein Patterning 1
Auxin responsive factor
Relative soil moisture content
Parallel analysis of RNA ends
Trichome birefringence-like protein
Early responsive dehydration-related.
The authors would like to thank Feng Chen for providing convenience for use of BGI Bio-Cloud Computing platform. This research was supported by the Hi-Tech Research and Development Program of China (2013AA102701), the National Natural Science Foundation of China (31070597, 31270656), the Ministry of Science and Technology of China (2009CB119101), and the Scientific Research and Graduate Training Joint Programs from BMEC (Stress Resistance Mechanism of Poplar).
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