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.
Populus trichocarpamicroRNADroughtTarget 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. trichocarpa leaves under control and drought conditions
According to a previous study on the relative soil moisture content (RSMC) of water-deficient soil [15, 28], P. trichocarpa plants were subjected to control levels (RSMC, 70–75%) and drought levels (RSMC, 15–20%). The two libraries were sequenced by an Illumina sequencer, yielding 27,333,282 reads for the control library (CL) and 30,806,496 reads for the drought library (DL) (Additional file 1: S1). After removing the low-quality sequences and adapter sequences, 26,229,957 clean sequences in CL and 30,233,516 clean sequences in DL were obtained, comprising 2,730,022 and 2,834,584 unique sequences, respectively (Table 1).
The size distribution of all unique sRNAs is summarized in Figure 1A. The displayed length of P. trichocarpa sRNA ranged from 16 to 27 nt, and the two major size classes were 24 nt (41.06% in CL and 43.94% in DL) and 21 nt (16.64% in CL and 16.05% in DL). This is in agreement with previous studies on sRNAs of P. trichocarpa  and M. truncatula  using high-throughput sequencing. To analyze the average abundance of each length between sRNAs of CL and DL further, we measured the ratio of raw and unique reads (Figure 1B). The redundancies of sRNAs varied widely in length, and the 20 and 21 nt sRNAs displayed the highest redundancies. The average ratio of redundant and unique sequences of sRNAs of the two libraries showed obvious changes in 21 nt sRNAs; the redundancy of DL was 37.16% greater than that of CL. This may be why drought stress strongly induced the expression of these 21 nt sRNAs; most conserved miRNAs belong to this group.
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
After identifying potential siRNAs and conserved miRNAs from the unique sRNA sequences, the remaining sRNA sequences were potential candidate miRNAs. For the identification of new miRNA, the primary criterion was a stable hairpin structure. After pooling the reads of the two libraries and analyzing the precursors of potential miRNAs using the MFOLD web server, we found 274 potential candidate miRNAs (Additional file 3: S3 and Table 2). In compliance with the plant miRNA criteria of Meyers , only 65 miRNAs with star sequences from 54 families were chosen as novel non-conserved miRNAs. Of these, 47 miRNAs were 21 nt long, eight miRNAs were 22 nt, five miRNAs were 20 nt and the remaining five were 19 nt long. The nucleotide bias at the first nucleotide showed a tendency to be U (41% of 65 novel non-conserved miRNAs). This would allow for easier miRNA RISC loading assisted by AGO protein  and is consistent with the trend of conserved miRNAs in plants .
New miRNAs inP. trichocarpa
‘LM’, Length of miRNAs. ‘nt’, neuleotides. ‘LP’, Length of precursors. ‘A’, refer to ; ‘B’, refer to ; ‘C’, refer to ; ‘D’, refer to ; ‘E’, refer to ; ‘F’, refer to .
These RNA structures were predicted by MFOLD software and manually checked according to the criteria of Meyers . The lowest minimum free energy (MFE) of all hairpin structures of the novel miRNAs precursors was −31.9 kcal/mol (Table 2), which is slightly lower than the threshold of −30 kcal/mol reported in a previous study . All precursors of novel miRNAs had regular hairpin structures (Additional file 4: S4), and four of these (Ptc-miRn5, Ptc-miRn11, Ptc-miRn38, and Ptc-miRn50) are presented in Figure 2.
Differential expression of miRNAs in P. trichocarpa
To identify drought-responsive miRNAs from P. trichocarpa, the number of normalized miRNA reads of CL and DL were compared. Based on the sequencing results, the differential expression of miRNAs greater than two-fold were chosen for experimental validation by quantitative real time polymerase chain reaction (RT-qPCR) (Additional file 5: S5). As shown in Figure 3, the expression patterns of the sequencing and RT-qPCR results of drought-responsive miRNAs were consistent, both indicating that four miRNAs (Ptc-miR159a-c, Ptc-miR472a, Ptc-miR472b, and Ptc-miR473a) were upregulated after drought treatment, and that five miRNAs (Ptc-miR160a-d, Ptc-miR164a-e, Ptc-miR394a/b-5p, Ptc-miR408, and Ptc-miR1444b-c) were downregulated by drought stress .
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).
The verification of miRNA targets would provide further evidence for the existence of new non-conserved miRNAs. To identify the miRNA targets, two parallel analyses of RNA ends (PARE) libraries were constructed for the P. trichocarpa degradome sequencing. In particular, the sRNA-cleaved mRNAs ligated by 5′ RNA adapters used for degradome sequencing acquired 23,326,117 and 34,398,368 reads (longer than 18 nt) in the mRNA libraries of the two treatments after removing redundancy; 108,593 and 234,316 unique reads could be matched to the P. trichocarpa genome (version 2.0) without mismatches (Additional file 7: S7) . Fifty-three conserved and 19 new miRNA-targeted transcript pairs were confirmed by degradome sequencing. The target transcripts were pooled and categorized into three classes with reference to Arabidopsis. Eleven pairs of miRNAs and their targets belonged to category I, which accounts for the most abundant sequence reads at the cleavage site. A total of 8 and 53 miRNAs and transcript pairs belonged to categories II and III, respectively. In addition, 13 target transcripts were predicted previously by either PopGenIE site or us (Table 3).
Targets ofP. trichocarpamiRNAs 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
‘Cleavage site’, the cleavage site location at the area of coding sequence; ‘Position penalty score’, the same penalty score as the prediction of new miRNA targets; ‘MFE’, minimum free energy of the stem loop structure. The bold font of transcripts indicates that they were identified by prediction.
Plant miRNAs have a strong propensity for target genes with important functions . According to the biological functions described by UniProt (http://www.uniprot.org/), these target transcripts can be grouped into nine categories. The majority of targets fall into the stress-response category, suggesting that these genes are drought-responsive (Table 4). Several other groups contain genes that regulate transcription, oxidative reduction, transport, and lipid metabolism. In this study, miR396 targeted a MYB transcription factor, and Ptc-miRn30 targeted an F-box family protein. The annotation of targets not only indicated some transcription factors and F-box proteins, but also some superoxide dismutases (SODs) and other proteins involved in glucose and lipid metabolism. A Cu-Zn SOD was targeted by Ptc-miRn49. All of these results indicate that miRNAs and their targets are reliable.
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
Although miRNAs have been shown to play an important role in the drought stress response of P. trichocarpa, little information on high-throughput sequencing of P. trichocarpa is available in this area. The present study on drought-responsive miRNAs from P. trichocarpa will improve the understanding of the drought response of this species. We identified nine conserved miRNAs and three novel miRNAs that show significant changes in response to drought stress. The results were confirmed by both high-throughput sequencing and RT-qPCR. To obtain more information, we compared the identified drought-responsive miRNAs with those identified in other studies (Table 5) [12–15, 17, 21, 25, 40–46]. MiR159 and miR164 have not yet found to be drought-responsive in Populus plants, except in this research. In addition, miR472, miR473, and miR1444 were found to be drought-responsive only in Populus plants, including in this study. The regulatory direction of four miRNAs (miR160, miR472, miR473 and miR408) was identical in P. tomentosa and our research, which might be due to their close genetic relationship.
MiRNAs responsive to drought stress in diverse plant species
‘↑’, upregulated; ‘↓’, downregulated; ‘↑&↓’, some members were upregulated, some members were downregulated.
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).
The following p-value formulae were used:
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.
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.
RNA-induced silencing complex
Small interfering RNA
Minimum free energy
Quantitative real time polymerase chain reaction
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).
College of Biological Sciences and Biotechnology, National Engineering Laboratory of Tree Breeding, Beijing Forestry University
Lee RC, Feinbaum RL, Ambros V: TheC. elegansheterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.Cell 1993,75(5):843–854.PubMedView Article
Llave C, Xie Z, Kasschau KD, Carrington JC: Cleavage of scarecrow-like mRNA targets directed by a class ofArabidopsismiRNA.Science 2002,297(5589):2053–2056.PubMedView Article
Pfeffer S, Zavolan M, Grasser FA, Chien MC, Russo JJ, Ju JY, John B, Enright AJ, Marks D, Sander C: Identification of virus-encoded microRNAs.Science 2004,304(5671):734–736.PubMedView Article
Kurihara Y, Watanabe Y: Arabidopsismicro-RNA biogenesis through Dicer-like 1 protein functions.P Natl Acad Sci USA 2004,101(34):12753–12758.View Article
Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function.Cell 2004,116(2):281–297.PubMedView Article
Baumberger N, Baulcombe DC: ArabidopsisARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs.P Natl Acad Sci USA 2005,102(33):11928–11933.View Article
Valdes-Lopez O, Arenas-Huertero C, Ramirez M, Girard L, Sanchez F, Vance CP, Reyes JL, Hernandez G: Essential role of MYB transcription factor: PvPHR1 and microRNA: PvmiR399 in phosphorus-deficiency signalling in common bean roots.Plant Cell Environ 2008,31(12):1834–1843.PubMedView Article
Guo HS, Xie Q, Fei JF, Chua NH: MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals forArabidopsislateral root development.Plant Cell 2005,17(5):1376–1386.PubMedView Article
Aukerman MJ, Sakai H: Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes.Plant Cell 2003,15(11):2730–2741.PubMedView Article
Puzey JR, Karger A, Axtell M, Kramer EM: Deep Annotation ofPopulus trichocarpamicroRNAs from Diverse Tissue Sets.PLoS One 2012,7(3):e33034.PubMedView Article
Chen L, Ren Y, Zhang Y, Xu J, Zhang Z, Wang Y: Genome-wide profiling of novel and conservedPopulusmicroRNAs involved in pathogen stress response by deep sequencing.Planta 2012,235(5):873–883.PubMedView Article
Liu HH, Tian X, Li YJ, Wu CA, Zheng CC: Microarray-based analysis of stress-regulated microRNAs inArabidopsis thaliana.RNA 2008,14(5):836–843.PubMedView Article
Zhou LG, Liu YH, Liu ZC, Kong DY, Duan M, Luo LJ: Genome-wide identification and analysis of drought-responsive microRNAs inOryza sativa.J Exp Bot 2010,61(15):4157–4168.PubMedView Article
Frazier TP, Sun GL, Burklew CE, Zhang BH: Salt and drought stresses induce the aberrant expression of microRNA genes in tobacco.Mol Biotechnol 2011,49(2):159–165.PubMedView Article
Li BS, Qin YR, Duan H, Yin WL, Xia XL: Genome-wide characterization of new and drought stress responsive microRNAs inPopulus euphratica.J Exp Bot 2011,62(11):3765–3779.PubMedView Article
Qin YR, Duan ZX, Xia XL, Yin WL: Expression profiles of precursor and mature microRNAs under dehydration and high salinity shock inPopulus euphratica.Plant Cell Rep 2011,30(10):1893–1907.PubMedView Article
Chen L, Ren Y, Zhang Y, Xu J, Sun F, Zhang Z, Wang Y: Genome-wide identification and expression analysis of heat-responsive and novel microRNAs inPopulus tomentosa.Gene 2012,504(2):160–165.PubMedView Article
Chen L, Zhang Y, Ren Y, Xu J, Zhang Z, Wang Y: Genome-wide identification of cold-responsive and new microRNAs inPopulus tomentosaby high-throughput sequencing.Biochem Biophys Res Commun 2012,417(2):892–896.PubMedView Article
Monclus R, Dreyer E, Villar M, Delmotte FM, Delay D, Petit JM, Barbaroux C, Thiec D, Brechet C, Brignolas F: Impact of drought on productivity and water use efficiency in 29 genotypes ofPopulus deltoidesxPopulus nigra.New Phytol 2006,169(4):765–777.PubMedView Article
Ren Y, Chen L, Zhang Y, Kang X, Zhang Z, Wang Y: Identification of novel and conservedPopulus tomentosamicroRNA as components of a response to water stress.Funct Integr Genomic 2012,12(2):327–339.View Article
Allen CD, Macalady AK, Chenchouni H, Bachelet D, McDowell N, Vennetier M, Kitzberger T, Rigling A, Breshears DD, Hogg EH: A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests.Forest Ecol Manage 2010,259(4):660–684.View Article
Hamanishi ET, Campbell MM: Genome-wide responses to drought in forest trees.Forestry 2011,84(3):273–283.View Article
Tuskan GA, DiFazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A: The genome of black cottonwood,Populus trichocarpa(Torr. & Gray).Science 2006,313(5793):1596–1604.PubMedView Article
Lu S, Sun YH, Chiang VL: Stress-responsive microRNAs inPopulus.Plant J 2008,55(1):131–151.PubMedView Article
Klevebring D, Street NR, Fahlgren N, Kasschau KD, Carrington JC, Lundeberg J, Jansson S: Genome-wide profiling ofPopulussmall RNAs.BMC Genom 2009, 10:620.View Article
Kozomara A, Griffiths-Jones S: miRBase: integrating microRNA annotation and deep-sequencing data.Nucleic Acids Res 2011, 39:D152-D157.PubMedView Article
Hasio T: Plant responses to water stress.Annu Rev Plant Physiol Plant Mol Biol 1973, 24:519–570.View Article
Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL: Novel and mechanical stress-responsive microRNAs inPopulus trichocarpathat are absent fromArabidopsis.Plant Cell 2005,17(8):2186–2203.PubMedView Article
Barakat A, Wall PK, Diloreto S, Depamphilis CW, Carlson JE: Conservation and divergence of microRNAs inPopulus.BMC Genom 2007, 8:481.View Article
Hamilton AJ, Baulcombe DC: A species of small antisense RNA in posttranscriptional gene silencing in plants.Science 1999,286(5441):950–952.PubMedView Article
Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ: Criteria for annotation of plant microRNAs.Plant Cell 2008,20(12):3186–3190.PubMedView Article
Mi SJ, Cai T, Hu YG, Chen Y, Hodges E, Ni FR, Wu L, Li S, Zhou H, Long CZ: Sorting of small RNAs intoArabidopsisargonaute complexes is directed by the 5 ’ terminal nucleotide.Cell 2008,133(1):116–127.PubMedView Article
Voinnet O: Origin, biogenesis, and activity of plant microRNAs.Cell 2009,136(4):669–687.PubMedView Article
Bonnet E, Wuyts J, Rouze P, Van de Peer Y: Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences.Bioinformatics 2004,20(17):2911–2917.PubMedView Article
Allen E, Xie Z, Gustafson AM, Carrington JC: MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants.Cell 2005,121(2):207–221.PubMedView Article
Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, Weigel D: Specific effects of microRNAs on the plant transcriptome.Dev Cell 2005,8(4):517–527.PubMedView Article
German MA, Luo SJ, Schroth G, Meyers BC, Green PJ: Construction of Parallel Analysis of RNA Ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome.Nat Protoc 2009,4(3):356–362.PubMedView Article
Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ: Endogenous siRNA and miRNA targets identified by sequencing of theArabidopsisdegradome.Curr Biol 2008,18(10):758–762.PubMedView Article
Reyes JL, Chua NH: ABA induction of miR159 controls transcript levels of two MYB factors duringArabidopsisseed germination.Plant J 2007,49(4):592–606.PubMedView Article
Sun GL, Stewart CN, Xiao P, Zhang BH: MicroRNA Expression Analysis in the Cellulosic Biofuel Crop Switchgrass (Panicum virgatum) under Abiotic Stress.PLoS One 2012.,7(3):
Ni ZY, Hu Z, Jiang QY, Zhang H: Overexpression of gma-MIR394a confers tolerance to drought in transgenicArabidopsis thaliana.Biochem Biophys Res Commun 2012,427(2):330–335.PubMedView Article
Kantar M, Unver T, Budak H: Regulation of barley miRNAs upon dehydration stress correlated with target gene expression.Funct Integr Genom 2010,10(4):493–507.View Article
Ferreira TH, Gentile A, Vilela RD, Costa GGL, Dias LI, Endres L, Menossi M: microRNAs associated with drought response in the bioenergy crop sugarcane (Saccharumspp.).PLoS One 2012,7(10):e46703.PubMedView Article
Barrera-Figueroa BE, Gao L, Wu ZG, Zhou XF, Zhu JH, Jin HL, Liu RY, Zhu JK: High throughput sequencing reveals novel and abiotic stress-regulated microRNAs in the inflorescences of rice.BMC Plant Biol 2012, 12:132.PubMedView Article
Trindade I, Capitao C, Dalmay T, Fevereiro MP, dos Santos DM: miR398 and miR408 are up-regulated in response to water deficit inMedicago truncatula.Planta 2010,231(3):705–716.PubMedView Article
Oh JE, Hong SW, Lee Y, Koh EJ, Kim K, Seo YW, Chung N, Jeong M, Jang CS, Lee B: Modulation of gene expressions and enzyme activities of methionine sulfoxide reductases by cold, ABA or high salt treatments inArabidopsis.Plant Sci 2005,169(6):1030–1036.View Article
Oh SK, Baek KH, Seong ES, Joung YH, Choi GJ, Park JM, Cho HS, Kim EA, Lee S, Choi D: CaMsrB2, Pepper Methionine Sulfoxide Reductase B2, Is a Novel Defense Regulator against Oxidative Stress and Pathogen Attack.Plant Physiol 2010,154(1):245–261.PubMedView Article
Guo XL, Wu YR, Wang YQ, Chen YM, Chu CC: OsMSRA4.1 and OsMSRB1.1, two rice plastidial methionine sulfoxide reductases, are involved in abiotic stress responses.Planta 2009,230(1):227–238.PubMedView Article
Romero HM, Berlett BS, Jensen PJ, Pell EJ, Tien M: Investigations into the role of the plastidial peptide methionine sulfoxide reductase in response to oxidative stress inArabidopsis.Plant Physiol 2004,136(3):3784–3794.PubMedView Article
Ma HS, Liang D, Shuai P, Xia XL, Yin WL: The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance inArabidopsis thaliana.J Exp Bot 2010,61(14):4011–4019.PubMedView Article
Herl V, Fischer G, Reva VA, Stiebritz M, Muller YA, Muller-Uri F, Kreis W: The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-OXO-Delta(4,5)-steroid 5 beta-reductase activity inArabidopsis thalianaL.Biochimie 2009,91(4):517–525.PubMedView Article
Jun JH, Ha CM, Nam HG: Involvement of the VEP1 gene in vascular strand development inArabidopsis thaliana.Plant Cell Physiol 2002,43(3):323–330.PubMedView Article
Tagami Y, Inaba N, Kutsuna N, Kurihara Y, Watanabe Y: Specific enrichment of miRNAs inArabidopsis thalianainfected with Tobacco mosaic virus.DNA Res 2007,14(5):227–233.PubMedView Article
Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY: Control of root cap formation by microRNA-targeted auxin response factors inArabidopsis.Plant Cell 2005,17(8):2204–2216.PubMedView Article
Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington JC: Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages.Plant J 2007,52(1):133–146.PubMedView Article
Hu RB, Qi GA, Kong YZ, Kong DJ, Gao QA, Zhou GK: Comprehensive Analysis of NAC Domain Transcription Factor Gene Family inPopulus trichocarpa.BMC Plant Biol 2010, 10:145.PubMedView Article
Jeong JS, Kim YS, Baek KH, Jung H, Ha SH, Do Choi Y, Kim M, Reuzeau C, Kim JK: Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions.Plant Physiol 2010,153(1):185–197.PubMedView Article
Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L: Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice.P Natl Acad Sci USA 2006,103(35):12987–12992.View Article
Lu S, Yang C, Chiang VL: Conservation and diversity of microRNA-associated copper-regulatory networks inPopulus trichocarpa.J Integr Plant Biol 2011,53(11):879–891.PubMedView Article
Ravet K, Danford FL, Dihle A, Pittarello M, Pilon M: Spatiotemporal analysis of copper homeostasis inPopulus trichocarpareveals an integrated molecular remodeling for a preferential allocation of copper to plastocyanin in the chloroplasts of developing leaves.Plant Physiol 2011,157(3):1300–1312.PubMedView Article
Guo YH, Yu YP, Wang D, Wu CA, Yang GD, Huang JG, Zheng CC: GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.New Phytol 2009,183(1):62–75.PubMedView Article
Bischoff V, Nita S, Neumetzler L, Schindelasch D, Urbain A, Eshed R, Persson S, Delmer D, Scheible WR: TRICHOME BIREFRINGENCE and its homolog AT5G01360 encode plant-specific DUF231 proteins required for cellulose biosynthesis inArabidopsis.Plant Physiol 2010,153(2):590–602.PubMedView Article
Gonzalez WL, Negritto MA, Suarez LH, Gianoli E: Induction of glandular and non-glandular trichomes by damage in leaves ofMadia sativaunder contrasting water regimes.Acta Oecol 2008,33(1):128–132.View Article
Bacelar EA, Correia CM, Moutinho-Pereira JM, Goncalves BC, Lopes JI, Torres-Pereira JMG: Sclerophylly and leaf anatomical traits of five field-grown olive cultivars growing under drought conditions.Tree Physiol 2004,24(2):233–239.PubMedView Article
Felippes FF, Weigel D: Triggering the formation of tasiRNAs inArabidopsis thaliana: the role of microRNA miR173.EMBO Rep 2009,10(3):264–270.PubMedView Article
Gong HJ, Chen KM, Zhao ZG, Chen GC, Zhou WJ: Effects of silicon on defense of wheat against oxidative stress under drought at different developmental stages.Biol Plantarum 2008,52(3):592–596.View Article
Sepulveda-Jimenez G, Rueda-Benitez P, Porta H, Rocha-Sosa M: A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress.J Exp Bot 2005,56(412):605–611.PubMedView Article
Tognetti VB, Van Aken O, Morreel K, Vandenbroucke K, de Cotte BV, De Clercq I, Chiwocha S, Fenske R, Prinsen E, Boerjan W: Perturbation of indole-3-butyric acid homeostasis by the UDP-Glucosyltransferase UGT74E2 modulatesArabidopsisarchitecture and water stress tolerance.Plant Cell 2010,22(8):2660–2679.PubMedView Article
Kannangara R, Motawia MS, Hansen NKK, Paquette SM, Olsen CE, Moller BL, Jorgensen K: Characterization and expression profile of two UDP-glucosyltransferases, UGT85K4 and UGT85K5, catalyzing the last step in cyanogenic glucoside biosynthesis in cassava.Plant J 2011,68(2):287–301.PubMedView Article
Chang S, Puryear J, Cairney J: A simple and efficient method for isolating RNA from pine trees.Plant Mol Biol Rep 1993, 11:113–116.View Article
Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H, Turner DJ: A large genome center’s improvements to the Illumina sequencing system.Nat Methods 2008,5(12):1005–1010.PubMedView Article
Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J: SOAP2: an improved ultrafast tool for short read alignment.Bioinformatics 2009,25(15):1966–1967.PubMedView Article
Gardner PP, Daub J, Tate J, Moore BL, Osuch IH, Griffiths-Jones S, Finn RD, Nawrocki EP, Kolbe DL, Eddy SR: Rfam: Wikipedia, clans and the “decimal” release.Nucleic Acids Res 2011, 39:D141-D145.PubMedView Article
Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ: miRBase: tools for microRNA genomics.Nucleic Acids Res 2008, 36:D154-D158.PubMedView Article
Zuker M: Mfold web server for nucleic acid folding and hybridization prediction.Nucleic Acids Res 2003,31(13):3406–3415.PubMedView Article
Audic S, Claverie JM: The significance of digital gene expression profiles.Genome Res 1997,7(10):986–995.PubMed
Man MZ, Wang X, Wang Y: POWER_SAGE: comparing statistical tests for SAGE experiments.Bioinformatics 2000,16(11):953–959.PubMedView Article
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method.Methods 2001,25(4):402–408.PubMedView Article
Mallory AC, Reinhart BJ, Jones-Rhoades MW, Tang GL, Zamore PD, Barton MK, Bartel DP: MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5 ’ region.EMBO J 2004,23(16):3356–3364.PubMedView Article
Brennecke J, Stark A, Russell RB, Cohen SM: Principles of MicroRNA-target recognition.PLoS Biol 2005,3(3):404–418.View Article
Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM: Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.Nature 2005,433(7027):769–773.PubMedView Article
Addo-Quaye C, Miller W, Axtell MJ: CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets.Bioinformatics 2009,25(1):130–131.PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.