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Article

Genome-Wide Identification of miR169 Family in Response to ABA and Salt Stress in Poplar

by
Ruiqi Wang
1,
Yuting Wang
1,
Yongmei Gu
2,
**yu Yan
1,
Wenna Zhao
1 and
Tingbo Jiang
1,*
1
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China
2
Key Laboratory Saline Alkali Vegetat Ecol Restorat, College of Life Science, Northeast Forestry University, Harbin 150040, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(5), 961; https://doi.org/10.3390/f14050961
Submission received: 24 February 2023 / Revised: 4 May 2023 / Accepted: 5 May 2023 / Published: 6 May 2023
(This article belongs to the Special Issue Advances in Ecological Genomics of Forest Trees)
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Abstract

:
The miR169 family is one of the largest families of known miRNAs, which performs important functions in plant growth, development, and responses to biotic/abiotic stresses. However, its functions in response to abiotic stresses are still unclear in poplar. In present study, a total of 33 precursor MIR169s were identified from poplar and divided into 3 groups by evolutionary analysis and multiple sequence alignment, with the members in same group sharing similar motifs. Collinearity analysis revealed miR169s in other species that are homologous to poplar miRNAs. Cis-acting elements predication showed that miR169s may respond to ABA (Abscisic acid) and salt stress, which was verified by qRT-PCR. In addition, 12 pairs of miR169/target gene modules were identified by degradome sequencing and most of these modules responded to ABA and salt stress. Specifically, a part of miRNAs showed opposite expression trends with their targets at a certain period, demonstrating a repressive effect on the target genes. All the results suggest that miR169s perform important functions in response to abiotic stresses in poplar.

1. Introduction

MicroRNAs (miRNAs) are noncoding RNAs with about 22–24 nucleotides in length, which perform important roles in post-transcriptional control by targeting complementary regions of mRNAs [1,2,3,4]. So far, several studies have shown that miRNAs perform a crucial role in the regulation of plant growth, development, and stress tolerance [5,6,7,8]. For instance, miR156 represses Arabidopsis adventitious root development by regulating target squamosa promoter binding protein-like (SPL) genes [9]. MiR319 could regulate TEOSINTE BRANCHED 1, cycloidea, and PCF-domain family protein 4 (TCP4), participating in the process of wood formation [10]. Among banana miRNAs, 113 known miRNAs and 26 novel banana-specific miRNAs were found to respond to temperature stress [11]. Peu-miR164s (Populus Euphratica, peu/pe) and their target gene, No apical meristem, ATAF1-2, and Cup-shaped cotyledon (PeNAC), were reported to exhibit sensitive responses to drought, salt, and ABA stress [12].
MiRNAs are evolutionarily conserved across plant species, and mature miRNAs show a high degree of similarity between species, which indicates their conserved functions across species. There are many miRNAs whose nucleic acid sequences are highly similar between family members, differing by only one base [13]. Compared to mature miRNAs, the similarity was low among different precursor miRNAs in the same family [13,14,15,16,17,18]. MiR169 family is one of the largest known miRNA families and has been identified from over 40 species [19,20]. For example, there are 15 miR169s in Arabidopsis thaliana, 25 in grape, and 32 in maize [21]. The ubiquitous presence of miR169s across plant species reflects the fact that it has an important biological function.
Previous studies have found that miR169s are closely associated with plant growth and developmental processes [22,23,24]. It has been reported that miR169defg indirectly affects lateral root initiation by regulating nuclear factor Y, subunit A2 (NF-YA2), in Arabidopsis [25]. MiR169 in Medicago truncatula was found to have an essential effect on the differentiation of nodule cells by regulating the expression of heme activator protein 2-1 (MtHAP2-1) [23]. ptc-MIR169 (Populus trichocarpa, ptr/ptc) was involved in regulating the timing of shoot dormancy in poplar [26]. MiR169 could also mediate early flowering in Arabidopsis under cold, salt, and drought stresses [27]. In addition, miR169s are reported to be involved in regulating plant responses to biotic stresses. For example, miR169 negatively regulates rice immunity against Magnaporthe oryzae by differentially repressing its target genes [28]. Over-expression of miR169o induces vulnerability of rice to bacterial blight [29]. On the other hand, miR169s also perform an important function in abiotic stresses. Salt stress can negatively affect the growth and development of most plants. Within minutes of a plant experiencing salt shock, water deficit, and wilting occur due to rapid changes in the osmotic pressure difference between the plant and the external environment [30,31]. Accompanying this water deficit stress are abscisic acid (ABA) biosynthesis and transportation throughout the plant initiating stomatal closure, among many other responses [32]. It has been shown that miR169s are able to respond to salt stress. For instance, most zma-miR169s (Zea mays, zma) and its targets in maize leaves can respond to polyethylene glycol (PEG, used to provide osmotic stress), ABA, and salt treatment [33]. Subsequently, ABA is an important plant hormone that regulates plant growth, development, and stress responses. It performs a vital role in a variety of physiological processes in plants, such as stomatal closure, cuticular wax accumulation, leaf senescence, bud dormancy, seed germination, osmotic regulation, and growth inhibition, among many others [34]. Endogenous ABA levels in plants can be fine-tuned by environmental factors (including salt, dehydration, drought, and other treatments) and are involved in the process of inducing plant resistance to these factors [34]. Treating plants with exogenous ABA can mimic the accumulation of endogenous ABA in plants under environmental stress, hel** to reveal the role of this hormone in the stress response. Previous studies have shown that miR169s are able to respond to ABA treatment and perform specific functions in different plant species. For example, the majority of zma-miR169s and their target genes were found to be able to respond to ABA treatment in maize [33]. MiR169 and its target HAP2-6 regulated by ABA are involved in poplar cambium dormancy [35]. Overall, simultaneous studies of miR169s responding to salt and ABA treatments provide great insight into the molecular mechanisms of plant stress resistance.
Populus trichocarpa was the first woody plant with a genome sequenced and has become a model plant in woody plant research [36]. There is a lack of research on the miR169 family in P. trichocarpa. In this study, a total of 33 precursor MIR169s/36 (the 3 letters of MIR are capitalized to indicate miRNA precursor [13]) mature miR169s members were identified from poplar using bioinformatics methods, and their expression patterns in different tissues under abiotic stresses were analyzed using qRT-PCR. The target genes of some miR169s were also obtained using degradome libraries, and their expression relationship under different stresses was also verified. This study provides new information about the evolutionary relationships of the miR169 family, which serves as a reference for understanding the biological functions of miR169s in poplar.

2. Materials and Methods

2.1. MIR169s Structure and Phylogenetic Analysis

Sequences of precursor and mature miR169s from Populus trichocarpa, Arabidopsis thaliana, and Vitis vinifera were obtained from miRBase (Release 22.1) and PMRD databases [37]. Clustal X2 [38] was used for multiple sequence alignment; due to the large differences between the sequences, the gap opening and gap extension are set to 10 and 0.2, respectively. MEGA X software 10.2 [39] was used to construct a UPGMA tree with the bootstrap value set to 10,000. MEME [40] was used to identify the motif compositions and distributions of miR169s. All those generated files were visualized using TBtools (version 0.665) [41] and Itools v6 (https://itol.embl.de/, accessed on 23 April 2023) software.

2.2. Chromosomal Localization and Collinearity Analysis

Precursor MIR169s were searched in Populus trichocarpa, Arabidopsis thaliana, and Vitis vinifera genomes and mapped on the Populus trichocarpa 4.1 genomic library (Phytozome 13, https://phytozome-next.jgi.doe.gov/, accessed on 10 January 2023) to obtain updated genomic coordinates. The threshold was set to ≥95% to obtain miRNAs with high homology. Then, the location of the resulting sequences was compared to all miRNA locations in these three species to determine colinear relationships. The resulting files were visualized with TBtools.

2.3. Cis-Acting Element Analysis

The promoter sequences of miR169 were obtained using Gtf/Gff Sequences Extract of TBtools. The online tool PlantCRAE [42] was used to extract cis-acting elements. The resulting component information was submitted to TBtools for visual analysis.

2.4. Prediction and Identification of miRNA Target Genes

Stand-alone version PatMatch software (Version 1.2) [43] and psRNATarget [44] were used to predict target genes of miR169s, with the intersection of the two prediction results as candidate target genes. The degradome (SRR8517155, SRR8517156, and SRR8517157) were analyzed to validate these candidate target genes. Clean reads obtained from the degradome library were used for target identification. The miRNA targets were scanned using CleaveLand 4.0 pipeline. All degradome clean reads were mapped to the transcript dataset, and scores were calculated based on the signature (abundance of potential slice ends based on the distribution of reads). The stricter thresholds were used to filter the targets, with Score ≤4.5 and Degradome Category ≤2. Functional annotation of the acquired target genes was performed using online software Omicshare tools (Version 3.0) (https://www.omicshare.com/, accessed on 22 January 2023).

2.5. Plant Treatments

The Poplus trichocarpa clone Nisqually-1 was used in this study [45]. The plantlets were planted in humus soil and grown under 16/8 hours (h) day–night photoperiod at 25 °C in the greenhouse. The different tissue samples were collected from 90 days-old poplar seedlings for gene expression analysis, the third to eighth functional leaves and stem segments and the intact roots were harvested for qPCR. Four-week-old seedlings grown in the same environment were selected for the ABA treatment. The leaves of the plants were sprayed with 100 µM ABA so that the leaves were covered with water droplets and did not fall off, while distilled water was used to spray the plants as a control. A total of 12 h later, at the same time, the functional leaves of control and treatment were collected. Plants grown for 90 days in the same environment were selected for salt treatment with a 200 mM NaCl solution. The soil was completely saturated with 1 L NaCl solution as the treatment group. A total of 1L of distilled water was used to water the plants as a control. Watering was performed again after 12 h. The functional leaves from the control and treatment groups were collected at the same time after 24 h. All samples had three biological replicates.

2.6. qRT-PCR Analysis

Total RNA was extracted using a MolPure® Plant Plus RNA kit (Yeasen, Shanghai, China). Hifair® miRNA 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China) was used to synthesize first-strand cDNA. Hieff® qPCR SYBR® Green Master Mix (Yeasen, Shanghai, China) was applied to identify the expression patterns of ptc-miR169s, and the 5.8S rRNA gene was used as an endogenous reference. For target genes, β-actin was selected as a reference gene for normalization. The 2−ΔΔCT method was used to analyze the relative expression changes of miRNAs [46]. All of the qPCR reactions were conducted with three replicates. Standard errors and standard deviations were calculated from three replicates.

3. Result

3.1. MIR169s Structure and Evolutionary Relationship

To identify miR169s in poplar, a phylogenetic tree was generated by searching miR169 precursor sequences from Populus trichocarpa, Vitis vinifera, and Arabidopsis thaliana, respectively. A total of 72 miR169s precursor sequences were obtained from miRbase [37] and PMRD databases [47], including 33 ptc-MIR169s from poplar, 14 ath-MIR169s (Arabidopsis thaliana, ath) from Arabidopsis, and 25 vvi-MIR169s (Vitis vinifera, vvi) from grape (Figure 1, Table S1). Based on high bootstrap values, MIR169s were divided into three groups, which contain 9 MIR169s, 28 MIR169s, and 35 MIR169s, respectively. As shown in Figure 1, all three subgroups cover MIR169s from three species. We also found that grape miR169s were always present on the branch where poplar MIR169s were located. This suggests that poplar MIR169s are evolutionarily more closely related to the MIR169s from grapes. Only ath-MIR169b and ath-MIR169h are on the same branch with grape and poplar; all other Arabidopsis MIR169s are on relatively separate branches. It was also found that the sequence lengths of Arabidopsis MIR169s were, on average, greater than 200 bp, with only ath-MIR169b and ath-MIR169f less than 200 bp. In contrast, the sequence lengths of poplar and grape MIR169s were mostly less than 200 bp, with only ptc-MIR169f, ptc-MIR169k, and vvi-MIR169y greater than 200 bp. This may be one reason that poplar miR169s are evolutionarily closer to grapes.
In addition, we analyzed the sequence structure of MIR169s in the three species and found that the sequences in mature regions were extremely similar across plant species, with the seed sequence of 18 bp in a mature region consisting of AGCCAAGGATGACTTGCC (Figure 2). The MIR169s with high similarity shared many same target genes, and the binding effect on target genes was strongly conserved across plant species [48,49,50]. To characterize the sequence structure of poplar MIR169s, ptc-MIR169s motifs were predicted using MEME software (Version 5.5.2), and a total of five conserved motifs were identified from ptc-MIR169s (Figure S1). Among them, motif 1 represents the mature body region of MIR169s, which appeared in all groups. Additionally, motif 2 appeared in all MIR169s except ptc-MIR169y; it contains miRNA precursor hairpin structure and the region complementary to mature regions. Motif 3, motif 4, and motif 5 only appeared in Group II. There are three MIR169s containing motif 3 in the same branch, including ptc-MIR169ab, ptc-MIR169ae, and ptc-MIR169af. There are also three ptc-MIR169s including ptc-MIR169ag, ptc-MIR169o, and ptc-MIR169p containing motif 4, which are also in the same branch. These suggest that the MIR169s in the two branches are evolutionarily stable. Motif 5 only appeared in five MIR169s in Group II, which also appeared in the closer branches. Overall, the miRNA precursors with the same motifs were shown to be conserved in evolutionary relationships.

3.2. Chromosomal Distribution and Collinearity Analysis of miR169s

We updated the chromosome location of miR169s according to the Populus trichocarpa v4.1 database in Phytozome (Table S2). To visualize the chromosome location of each ptc-miR169s, we mapped miRNA distribution based on their starting position on the chromosome (Figure 3a). The results showed that there were 36 miR169s not evenly distributed on the 13 chromosomes except chromosomes 2, 4, 11, 12, 14, and 16 (Figure 3a). Among them, chromosomes 1 and 18 contained the highest number of ptc-miR169s, and other chromosomes contained 1–4 ptc-miR169s.
To compare sequence similarity among miRNAs in poplar, we searched all miRNAs in the poplar genome using blastn, and deeply filtered the searched fragments, matched known miRNAs based on positional information, and finally obtained the intra-species collinearity map of poplar miRNAs (Figure 3a, Table S3). Of the 34 homologous pairs of ptc-miR169s, we found six miRNAs in chromosomes 1, 5, and 7 displaying three pairs of tandem duplication events. Additionally, 31 pairs of ptc-miR169s exhibited segmental duplication on different chromosomes (Figure 3a). The overall results suggest that segmental duplication performs an important role in miR169 amplification. To investigate miR169s genomic duplication events between poplar and two other species (Arabidopsis thaliana and Vitis vinifera), we obtained two collinearity maps, including ptc-ath and ptc-vvi (Figure 3b, Table S3). A total of 62 and 48 colinear pairs were found in ptc-ath and ptc-vvi, respectively. As many as 19 of the 33 ptc-miR169s had colinear miRNAs from other species, and 10 of these 19 miRNAs were found to have colinear miRNAs in both Arabidopsis and grape, including ptc-miR169a, ptc-miR169b-5p, ptc-miR169c~h, ptc-miR169p, and ptc-miR169s. Overall, miR169s were highly conserved across these three species, which is consistent with the highly conserved features of miRNAs across plant species.

3.3. Cis-Elements Analysis of miR169 Promoters

We predicted the cis-elements in the 2000 bp upstream sequence of all ptc-miR169s through PLANTCARE. A total of 885 cis-elements were detected in the promoter regions of miR169s (Table S4). Except for core promoters and enhancers, light responsive elements were the most diverse and numerous, with 368 of 26 types (Figure 4). It mainly includes 63 light-induced stem- and leaf-specific expression promoter G-boxes, 44 photosynthetic elements GT1-motifs, 24 light-responsive GATA-motifs, etc. This was followed by hormone response elements, with 326 of 11 types (Table S4), including 99 abscisic acid response elements (ABRE), 70 methyl jasmonate response elements (CGTCA-motif, TGACG-motif), 22 gibberellin response elements (GARE-motif, P-box, TATC-box), 55 salicylic acid response elements (TCA-element, as-1), 19 auxin response elements (TGA-element, AuxRR-core), and 61 estrogen response elements (ERE). Six types of biotic/abiotic stress response elements were identified (Table S4), including 53 damage and pathogen response elements (W-box), 20 drought response elements (MBS), 17 low-temperature response elements (LTR), 16 defense and stress response elements (TC-rich repeats), and 13 dehydration response elements (DRE1, DRE core). In addition, ABRE, DRE1, DRE core, GT1-motif, and W box are known to be associated with salt stress response based on previous studies [51]. A total of 62 growth and development response elements were also identified in all promoters of ptc-miR169s (Table S4), divided into 6 types, including 20 O2 sites participating in zein metabolism regulation, 10 circadian involved in circadian control, 13 CAT-box related to meristem expression, 5 MBSI involved in flavonoid biosynthetic genes regulation, 8 GCN4_motif involved in endosperm expression, and 6 HD-Zip 1 participating in the differentiation of the palisade mesophyll cells. These results suggest that ptc-miR169s are widely involved in various biological processes, such as stress responses and plant growth and development.

3.4. Tissue-Specific Expression of miR169s in Poplar

To investigate the expression pattern of miR169s in poplar, three tissues (Root, Stem, and Leaf) were sampled from Populus trichocarpa for miR169s quantitative assays. Based on the expression levels of miRNAs, the miR169s could be clustered into two groups (Figure 5), which were highly expressed in root and leaf, respectively. The two miRNAs that were highly expressed in roots are ptc-miR169k/m and ptc-miR169s. Of these, miR169k/m was up-regulated by 166-fold compared to control (The expression of ptc-miR169u-5p in the roots was set to 1 as control.) and miR169s was up-regulated by 5-fold (Table S5). All remaining miRNAs were highly expressed in the leaves, with miR169d being the highest expression level of 1344-fold. This was followed by miR169y and miR169ab with 352- and 355-fold, respectively (Table S5). Overall, miR169s were most highly expressed in leaves and partially in roots, suggesting that they are likely to perform biological functions in roots or leaves.

3.5. Expression Pattern of miR169s under ABA and Salt Treatments

The promoter regions of miR169s contain a large number of ABA and salt response elements (Figure 4, Table S4), which indicates that miR169s are likely to perform a function in ABA and salt responses. Therefore, we analyzed the changes in the expression levels of miR169s in poplar under ABA and salt treatments. Compared to the control, the results showed that eight miR169s were significantly up-regulated, and four were significantly down-regulated under ABA treatment (Figure 6a). The expression of miR169n-5p was the most significant, with 2-fold up-regulation. While the expression of miR169y was the most significantly down-regulated, at 0.18-fold (Table S6). Under salt stress, most miR169 showed significantly up-regulated expression compared to the control, with only miR169u-5p showing no response to salt stress (Figure 6b). Among the miR169s, miR169s, miR169d, and miR169a were most significantly responding to salt stress, with 24-fold, 19-fold, and 17-fold upregulation, respectively (Table S7). Overall, among the miR169s, 12 miR169s were involved in response to ABA and 17 in response to salt treatment, suggesting that miR169s may perform some function during abiotic stresses.

3.6. Identification of miR169s Target Genes

As known, miRNAs generally perform biological functions by regulating downstream genes. Therefore, we explored downstream target genes of miR169s. PatMatch software (Version 1.2) and psRNATarget were used to obtain two sets of predictions, respectively, and their intersections were regarded as candidate targets. In order to validate the candidate targets, we cited “degradome” libraries for searching targets of miR169 in P. trichocarpa. The thresholds of Score ≤4.5 and Degradome Category ≤2 were used to filter the degradome data (Table 1). After removing the differential transcripts, we finally obtained 12 miRNA/target gene modules, namely, miR169aa/ATVHA-C, miR169ag/PtrNFYA1, miR169ag/PtrNFYA2, miR169k/PtrNFYA7, miR169m/PtrNFYA7, miR169o/PtrNFYA6, miR169o/NPH3, miR169q/NDL2, miR169x/PtrNFYA7, miR169y/PtrNFYA3, miR169y/PtrNFYA6, and miR169y/PtrNFYA1 (Table 1). Based on gene function annotation, we found that most of the target genes in these modules are from NFYA transcription factor family. Previous studies demonstrated that various members of the NFYA family can respond to salt and ABA treatments, which may perform important functions in abiotic stresses [35,52].

3.7. Expression Pattern of miR169s with Their Target Genes

We analyzed the expression patterns of the target genes in the modules under ABA and salt treatment (Figure 6). The results showed that all the target genes of miR169s could significantly respond to ABA and salt treatments (Figure 6, Tables S6 and S7). Generally, miRNA inhibits the expression of target genes by cutting off their mRNA chain after complementary binding with the target gene; thus, miRNAs and target genes usually show opposite expression trends [53,54]. Accordingly, we performed Pearson correlation coefficient analysis on miR169s and their target genes, screening for miRNA/target modules that express negative correlations after stress. The results showed (Table 2, Figure 7) that miRNAs in a total of six modules (miR169aa/ATVHA-C, miR169ag/PtrNFYA2, miR169x/PtrNFYA7 and miR169y/PtrNFYA1/3/6) were significantly negatively correlated (correlation ≤ 0.8, p-value ≤ 0.05) with target genes under ABA treatment. Additionally, miRNAs in all modules were significantly negatively correlated with target genes under salt stress (Table 2, Figure 7). Overall, these negative correlation modules may be important regulators of ABA and salt responses in poplar.

4. Discussion

MiR169 is a functionally important miRNA family in plants, and almost all plants contain miR169s [17,28,33,35,55]. In our study, a total of 33 precursors of miR169 were found in poplar, 25 in grape, and 14 in Arabidopsis. The average sequence length of Arabidopsis MIR169s was significantly longer than that of poplar and grape MIR169s (Figure 1, Table S1). Moreover, poplar and grape had more MIR169s in the same branch compared to Arabidopsis. Overall, this suggests that poplar and grape are evolutionarily closer. Multiple sequence comparisons of MIR169s from poplar, grape, and Arabidopsis strongly support they are conserved in plants. In addition, the 5′ end of the mature region is much more similar compared to the other regions, which determines the type of AGO that attached upon maturation, and, in turn, affects its function [56]. The specificity of the 3′ end of the miRNA may be a factor in regulating the activity and stability of different miRNAs [17,57]. We found an identical motif at the 3′ end of all ptc-MIR169s, suggesting that the activity and stability of ptc-miR169s may be similar (Figure S1).
As we know, gene duplication and random hairpin formation are two main sources of new miRNAs [58]. Gene duplication is the main pattern of gene family expansion and includes tandem duplication and segmental duplication events [58,59,60]. Collinearity analysis revealed that although most ptc-miR169s appeared in clusters, tandem duplication events occurred in only 3 pairs, and segmental duplication events occurred in 31 pairs. This indicates that segmental duplication performs a major role in the expansion of miR169 family in poplar. By cross-species collinearity analysis, we also identified 62 and 48 colinear miRNAs in Arabidopsis and grape, respectively, with high homology (≥95%). According to reports, nearly 20% of plant miRNAs are clustered and generally contain the same conserved miRNAs [61]. These clustered miRNAs generally expand as chromosomes evolve [61].
Analysis of promoter cis-elements revealed that the promoters of ptc-miR169s contain a large number of ABA and salt response elements. This suggests that miR169s may be involved in response to abiotic stresses. Subsequent experimental validation indicated that 12 miR169s were found to be responsive to ABA and 17 to salt. It is well known that plants experiencing salt stress may be accompanied by endogenous ABA biosynthesis [32,62], but plant responses to salt or other stresses are not entirely dependent on the ABA pathway. In the study, eight miR169s were significantly up-regulated under both salt and ABA treatments compared to the control, namely ptc-miR169d, ptc-miR169k/m, ptc-miR169s, ptc-miR169o, ptc-miR169p, ptc-miR169ab, ptc-miR169ag, and ptc-miR169q. It implies that these eight miR169s may respond to salt stress by increasing endogenous ABA content in plants. The other nine salt stress-responsive miR169s may function independently of the ABA pathway. To further investigate the function of these miRNAs, we obtained miRNA/target modules that are negatively correlated in ABA and salt treatment by Pearson’s correlation coefficient. In ABA treatment, miRNAs in six modules were negatively correlated with the expression of their targets. In salt stress, all identified modules were negatively correlated. This indicates that the miRNAs in the 12 modules do have regulatory effects on the targets during ABA and salt treatments. Previous reports have shown that the expression levels of miRNAs are negatively correlated with those of target genes in the short term (4–48 h), which is a typical pattern of regulation [33]. This coincides with the results we obtained. In addition, miR169ag belongs to one of the miR169 due to the ABA pathway in response to salt stress (Figure 6). miR169ag and its target NFYA2 were significantly negatively correlated in both ABA and salt treatments, suggesting that the miR169ag/NFYA2 module may have an important function in the ABA pathway under salt stress.
The regulation of downstream target genes is the main mode in which miRNAs exercise their functions. Therefore, the exploration of miRNA functions is dominated by the study of miRNA/target gene modules. Of the modules we identified, miR169o/NFYA6 has been reported in poplar. Overexpression lines of miR169o showed significant drought resistance in P. trichocarpa, while overexpression lines of its target gene NFYA6 showed reduced resistance to drought. Thus the enhanced drought resistance of miR169o overexpression may be due to its inhibition of the downstream drought-sensitive gene NFYA6 [63]. In poplar (Populus alba × P. gllandulosa cv. “84 k”, Pag) studies, overexpression of PagHAP2-6/NFYA6 increased plant resistance to exogenous ABA, and PagHAP2-6 was verified to be cleaved by Pag-miR169a [34]. In addition, the miR169/NFYA module has also been reported to be involved in abiotic responses in other plant species. miRNA169-target NFYA5 responds to drought, and ABA in Arabidopsis, and overexpression of AtNFYA5 (homologous with PtrNFYA3) improves drought resistance in Arabidopsis [64]. The miR169/NFYA module was also found to be involved in ABA response in maize, suggesting that these modules are functionally similar and conserved across plant species [33,65]. In addition, the miR169-NFYA module was involved in ABA pathway-dependent drought resistance in cereals [66]. In the study of Malus hupehensis Rehd. var. **yiensis Jiang (**yi Tiancha, PYTC), the mhp-miR169/NFYA module was found to be involved in the salt resistance of roots [67]. zma-miR169 and their target ZmNF-YA genes were reported to respond positively to salt stress in maize leaves [33]. Overall, the miR169/NFYA module is actively involved in abiotic stress-related biological processes. The 12 miR169/target modules in the study have been preliminarily shown to respond positively to ABA and salt treatments, providing a basis for subsequent studies in poplar.

5. Conclusions

In this study, we identified 33 ptc-miR169 precursors and 36 mature miRNAs in poplar. The MIR169s were divided into three clusters by evolutionary analysis, and the seed sequences of the mature region of the MIR169s were identified by multiple sequence alignment. Collinearity analysis revealed poplar miR169s were homologous to the miRNAs in other plant species, such as grape and Arabidopsis. Cis-elements analysis in the promoters showed that miR169s were highly likely to be involved in ABA and salt response, which was verified by qPCR. On the other hand, 12 pairs of miR169/target modules were identified by degradome sequencing. Most of these modules responded to ABA, drought, and salt treatments, and a part of miRNAs showed opposite expression trends with their targets at a certain period, demonstrating their repressive effect on target genes. In conclusion, the study provides a reference for understanding the biological functions of miR169s in poplar.

Supplementary Materials

The following supporting information can be downloaded at: https://mdpi.longhoe.net/article/10.3390/f14050961/s1, Figure S1: Distribution of the conserved motifs in MIR169s analyzed by MEME; Table S1: Precursor sequences and lengths of Populus trichocarpa, Vitis vinifera, and Arabidopsis thaliana in miR169s; Table S2: Coordinates of ptc-miR169s in the Populus trichocarpa 4.1 genome; Table S3: The homologous relationships of miR169s in poplar themselves and poplar with other species; Table S4: The list of cis-elements of ptc-MIR169s promoter; Table S5: The tissue-specific expression patterns of ptc-miR169s as determined by qPCR analysis; Table S6: The expression pattern analysis ptc-miR169s and their targets under ABA stress in 0 h and 12 h by qPCR; Table S7: The expression pattern analysis ptc-miR169s and their targets under salt stress in 0 h and 24 h by qPCR; Table S8: The primers sequences used in this study.

Author Contributions

R.W. and T.J.: Designed research, Funding acquisition, Methodology. Y.W. and Y.G.: Conducted the experiments, Analyzed the data and wrote the manuscript. W.Z. and P.Y.: Cultured the plant material. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Major Project of Agricultural Biological Breeding (2022ZD0401504) and the Fundamental Research Funds for the Central Universities (2572018CL03).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of MIR169s in Populus trichocarpa (ptc), Vitis vinifera (vvi), and Arabidopsis thaliana (ath). 10,000 bootstrap replicates were applied to draw a UPGMA tree by MEGA X software 10.2. The bars represent the length of the nucleic acid sequence, the blue dashed lines represent 0 bp, the red dashed lines represent 200 bp, and the green dashed lines represent 400 bp. Black circles indicate ath-miR169s. Black rectangles indicate vvi-miR169s. Black stars indicate ptc-miR169s. The cyan background miRNA is classified as Group I, the pink background as Group II, and the dark green background as Group III.
Figure 1. Phylogenetic analysis of MIR169s in Populus trichocarpa (ptc), Vitis vinifera (vvi), and Arabidopsis thaliana (ath). 10,000 bootstrap replicates were applied to draw a UPGMA tree by MEGA X software 10.2. The bars represent the length of the nucleic acid sequence, the blue dashed lines represent 0 bp, the red dashed lines represent 200 bp, and the green dashed lines represent 400 bp. Black circles indicate ath-miR169s. Black rectangles indicate vvi-miR169s. Black stars indicate ptc-miR169s. The cyan background miRNA is classified as Group I, the pink background as Group II, and the dark green background as Group III.
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Figure 2. Nucleotide sequence structure analysis of MIR169s in Populus trichocarpa (ptc), Vitis vinifera (vvi), and Arabidopsis thaliana (ath). The bar represents consensus nucleotide abundance. The tree shown in this figure is the same as in Figure 1.
Figure 2. Nucleotide sequence structure analysis of MIR169s in Populus trichocarpa (ptc), Vitis vinifera (vvi), and Arabidopsis thaliana (ath). The bar represents consensus nucleotide abundance. The tree shown in this figure is the same as in Figure 1.
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Figure 3. Chromosomal localization and collinearity analysis of ptc-miR169s. (a) Chromosomes distribution of ptc-miR169s. The red lines represent pairs of tandem repeated genes, and the gray lines represent pairs of segmentally repeated genes. ptc represents Populus trichocarpa. (b) Cross-species collinearity analysis of miR169s. The pale orange lines represent collinear pairs of Populus trichocarpa (Pt) and Vitis vinifera (Vv), while the jade-green lines indicate collinear pairs of Populus trichocarpa (Pt) and Arabidopsis thaliana (At). The brown and dark green lines represent the colinear pairs of ptc-miR169s with vvi-miR169s and ath-miR169s, respectively. Ch represents chromosomes.
Figure 3. Chromosomal localization and collinearity analysis of ptc-miR169s. (a) Chromosomes distribution of ptc-miR169s. The red lines represent pairs of tandem repeated genes, and the gray lines represent pairs of segmentally repeated genes. ptc represents Populus trichocarpa. (b) Cross-species collinearity analysis of miR169s. The pale orange lines represent collinear pairs of Populus trichocarpa (Pt) and Vitis vinifera (Vv), while the jade-green lines indicate collinear pairs of Populus trichocarpa (Pt) and Arabidopsis thaliana (At). The brown and dark green lines represent the colinear pairs of ptc-miR169s with vvi-miR169s and ath-miR169s, respectively. Ch represents chromosomes.
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Figure 4. Predicted cis-elements in the promoter regions of ptc-miR169s. Different elements were indicated by different shapes; rectangles indicate phytohormone response elements, boxes indicate growth and development-related elements, ellipses indicate stress response related elements, circles indicate light response elements, and different elements are indicated by different colors. ptc represents Populus trichocarpa. The element counts are shown in Table S4. The tree shown in this figure is the same as in Figure 1.
Figure 4. Predicted cis-elements in the promoter regions of ptc-miR169s. Different elements were indicated by different shapes; rectangles indicate phytohormone response elements, boxes indicate growth and development-related elements, ellipses indicate stress response related elements, circles indicate light response elements, and different elements are indicated by different colors. ptc represents Populus trichocarpa. The element counts are shown in Table S4. The tree shown in this figure is the same as in Figure 1.
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Figure 5. The tissue-specific expression patterns of ptc-miR169s by qPCR analysis. 5.8S rRNA was used as a reference gene. The expression of ptc-miR169u-5p in root was set to 1. The data was processed using 2−ΔΔCt method. ptc represents Populus trichocarpa.
Figure 5. The tissue-specific expression patterns of ptc-miR169s by qPCR analysis. 5.8S rRNA was used as a reference gene. The expression of ptc-miR169u-5p in root was set to 1. The data was processed using 2−ΔΔCt method. ptc represents Populus trichocarpa.
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Figure 6. The expression pattern of ptc-miR169s under ABA (100 µM) and salt (200 mM) treatments. (a) and (b) represent the expression profile of miR169s in ABA and salt treatment, respectively. Control indicates that water treatment is performed in the same way. The data was processed using 2−ΔΔCt method. 5.8S rRNA was used as a reference gene. Asterisks indicate significant differences (t test, * p < 0.05, ** p < 0.01). ptc represents Populus trichocarpa.
Figure 6. The expression pattern of ptc-miR169s under ABA (100 µM) and salt (200 mM) treatments. (a) and (b) represent the expression profile of miR169s in ABA and salt treatment, respectively. Control indicates that water treatment is performed in the same way. The data was processed using 2−ΔΔCt method. 5.8S rRNA was used as a reference gene. Asterisks indicate significant differences (t test, * p < 0.05, ** p < 0.01). ptc represents Populus trichocarpa.
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Figure 7. Comparative analysis of expression patterns of miR169s and their target genes under ABA and salt treatments. (a–f) and (g–n) represent the expression patterns of miRNAs and their target genes in response to ABA and salt treatment, respectively. Control indicates that water treatment is performed in the same way. The left y-axis represents the relative expression of miRNA, and the right y-axis represents the relative expression of the target gene. Lines with nodes represent miRNAs. Bars represent target genes. 5.8S rRNA and β-actin was used as reference genes of miRNAs and target genes, respectively. Asterisks indicate significant differences with ABA treatment (t test, * p < 0.05, ** p < 0.01). ptc/ptr represents Populus trichocarpa.
Figure 7. Comparative analysis of expression patterns of miR169s and their target genes under ABA and salt treatments. (a–f) and (g–n) represent the expression patterns of miRNAs and their target genes in response to ABA and salt treatment, respectively. Control indicates that water treatment is performed in the same way. The left y-axis represents the relative expression of miRNA, and the right y-axis represents the relative expression of the target gene. Lines with nodes represent miRNAs. Bars represent target genes. 5.8S rRNA and β-actin was used as reference genes of miRNAs and target genes, respectively. Asterisks indicate significant differences with ABA treatment (t test, * p < 0.05, ** p < 0.01). ptc/ptr represents Populus trichocarpa.
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Table
Table 1. Target genes of known miRNAs validated with degradome sequencing.
Table 1. Target genes of known miRNAs validated with degradome sequencing.
miRNATranscriptCleave SiteScoreDegradome
Category		SymbolDescription
ptc-miR169aaPotri.017G061100.110994.502ATVHA-Cvacuolar ATP synthase subunit C (VATC)/V-ATPase C subunit/vacuolar proton pump C subunit (DET3)
ptc-miR169agPotri.009G060600.2135530PtrNFYA1nuclear factor Y
ptc-miR169agPotri.018G064700.1127820PtrNFYA2nuclear factor Y
ptc-miR169agPotri.018G064700.2133420PtrNFYA2nuclear factor Y
ptc-miR169agPotri.018G064700.396220PtrNFYA2nuclear factor Y
ptc-miR169kPotri.006G201900.49784.500PtrNFYA7nuclear factor Y
ptc-miR169mPotri.006G201900.315234.500PtrNFYA7nuclear factor Y
ptc-miR169oPotri.016G090400.1152842NPH3Phototropic-responsive NPH3 family protein
ptc-miR169oPotri.006G145100.2131310PtrNFYA6nuclear factor Y
ptc-miR169oPotri.006G145100.3136010PtrNFYA6nuclear factor Y
ptc-miR169oPotri.006G145100.4143310PtrNFYA6nuclear factor Y
ptc-miR169qPotri.018G054900.265330NDL2N-MYC downregulated-like 2
ptc-miR169qPotri.018G054900.154232NDL2N-MYC downregulated-like 2
ptc-miR169xPotri.006G201900.213454.500PtrNFYA7nuclear factor Y
ptc-miR169xPotri.006G201900.113634.501PtrNFYA7nuclear factor Y
ptc-miR169yPotri.001G266000.4117340PtrNFYA3nuclear factor Y
ptc-miR169yPotri.001G266000.5168940PtrNFYA3nuclear factor Y
ptc-miR169yPotri.009G060600.1126240PtrNFYA1nuclear factor Y
ptc-miR169yPotri.009G060600.6146340PtrNFYA1nuclear factor Y
ptc-miR169yPotri.001G266000.1121442PtrNFYA3nuclear factor Y
ptc-miR169yPotri.001G266000.2128642PtrNFYA3nuclear factor Y
ptc-miR169yPotri.001G266000.3133242PtrNFYA3nuclear factor Y
ptc-miR169yPotri.006G145100.1139030PtrNFYA6nuclear factor Y
ptc-miR169yPotri.006G145100.670030PtrNFYA6nuclear factor Y
Table
Table 2. Pearson correlation coefficient of miR169s and their target genes under ABA and salt treatment.
Table 2. Pearson correlation coefficient of miR169s and their target genes under ABA and salt treatment.
miRNATarget GeneABA TreatmentSalt Treatment
Correlationp-ValueCorrelationp-Value
ptc-miR169aaATVHA-C−0.975150.000919−0.986370.000278
ptc-miR169agPtrNFYA10.9658870.001726−0.989360.000169
ptc-miR169agPtrNFYA2−0.839370.03663−0.988630.000193
ptc-miR169k/mPtrNFYA70.9248870.008251−0.973510.001043
ptc-miR169oPtrNFYA60.9484450.003918−0.985570.000311
ptc-miR169oNPH30.7801790.067171−0.949220.003802
ptc-miR169qNDL20.9758020.000871−0.945940.004304
ptc-miR169xPtrNFYA7−0.885180.019017−0.946420.004229
ptc-miR169yPtrNFYA3−0.959440.002434−0.971520.001205
ptc-miR169yPtrNFYA6−0.880510.020562−0.93850.005558
ptc-miR169yPtrNFYA1−0.890410.017356−0.97390.001013
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Wang, R.; Wang, Y.; Gu, Y.; Yan, P.; Zhao, W.; Jiang, T. Genome-Wide Identification of miR169 Family in Response to ABA and Salt Stress in Poplar. Forests 2023, 14, 961. https://doi.org/10.3390/f14050961

AMA Style

Wang R, Wang Y, Gu Y, Yan P, Zhao W, Jiang T. Genome-Wide Identification of miR169 Family in Response to ABA and Salt Stress in Poplar. Forests. 2023; 14(5):961. https://doi.org/10.3390/f14050961

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Wang, Ruiqi, Yuting Wang, Yongmei Gu, **yu Yan, Wenna Zhao, and Tingbo Jiang. 2023. "Genome-Wide Identification of miR169 Family in Response to ABA and Salt Stress in Poplar" Forests 14, no. 5: 961. https://doi.org/10.3390/f14050961

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