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Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco
GENE-40731; No. of pages: 7; 4C: Gene xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco Bi-fei Wu a, Wan-feng Li b, Hai-yan Xu b, Li-wang Qi b, Su-ying Han a,⁎ a b
Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
a r t i c l e
i n f o
Article history: Received 23 April 2015 Received in revised form 5 July 2015 Accepted 22 July 2015 Available online xxxx Keywords: Cin-miR2118 TIR-NBS-LRR Caragana intermedia Drought tolerance
a b s t r a c t The miR2118 is highly conserved in leguminous plants. Its function is to regulate the expression of genes encoding the TIR-NBS-LRR resistance protein. In this study, cin-miR2118 from Caragana intermedia was functionally characterized, especially with regard to its role in drought stress resistance. Two target genes of cin-miR2118 were predicted and cloned, the occurrence of miR2118 target sequence in both genes indicated that they might be targets of cin-miR2118. We investigated the expression patterns of cin-miR2118 and its target genes in C. intermedia stems and found diverse changes in expression in response to drought stress. CiDR1 was negatively correlated with corresponding miR2118 expression while CiDR2 was positively correlated with cin-miR2118. For further study, induced tolerance was observed in the transgenic Tobacco with overexpression cin-miR2118 upon 140-min water deficiency. And the expression level of cin-miR2118 was dramatically increased under drought stress. These results reveal that cin-miR2118 exert positive effects on drought stress tolerance. In addition, our study unexpectedly found that overexpression of cin-miR2118 in Tobacco can cause phenotype changes, which suggested that cin-miR2118 may have a novel function as a growth regulator in Tobacco. © 2015 Published by Elsevier B.V.
1. Introduction MicroRNAs (miRNAs) are ~ 21 nt endogenous RNAs that are highly conserved among higher plants (Chen and Rajewsky, 2007; Jones-Rhoades et al., 2006). They use base-pairing interactions to regulate mRNA expression (Bushati and Cohen, 2007) by cleaving the targeted transcript, or translational inhibition following imperfect pairing. Although both mechanisms have been observed in plants, cleavage is the more common (Arenas-Huertero et al., 2009; Brodersen et al., 2008). Numerous recent studies have shown that miRNAs and their targets play important roles in multiple biological processes, such as plant growth (Guo, 2005), organ development (Emery et al., 1768–1774; Kim et al., 2005), signal transduction (Wang et al., 2004), pathogen infection (Prokhnevsky and Chapman, 2004), and stress responses (Sunkar, 2001–2019). With the rapid development of molecular biology techniques, many novel miRNAs have been identified, although little is known about their functions or target genes (Sunkar, 2001–2019; He and Hannon, 2004). Some miRNAs have been confirmed experimentally to be involved in a variety of abiotic stress responses, encompassing drought, salinity,
Abbreviations: C. intermedia, Caragana intermedia; miRNA, microRNA; NCBI, National Center for Biotechnology Information; Pre, precursor; qRT-PCR, quantitative reverse transcription PCR; RLM-5′RACE, RNA ligase-mediated amplification of cDNA ends. ⁎ Corresponding author. E-mail address: [email protected] (S. Han).
extreme temperatures, nutrition stress, ultraviolet (UV)-B radiation, and oxidative stress. miR398 was the first miRNA reported to respond to abiotic stress. miR398-directed mRNA cleavage of target genes CSD1 and CSD2 fine-tunes their expression under oxidative stresses (Sunkar, 2006), while over-expressing osa-miR396c in rice and Arabidopsis reduced salt and alkali stress tolerance (Gao et al., 2010). Other studies found that miR399 is strongly induced by low phosphate stress, and that it controls phosphate homeostasis by regulating the expression of a putative ubiquitin-conjugating E2 enzyme, UBC24, in Arabidopsis (Fujii et al., 2005). miR169g and miR169n, which both target NF-YA, exhibited overlapping and distinct responses to drought and salt stresses (Zhao et al., 2009). Additionally, Zhou identified that 21 miRNA genes in 11 miRNA families are up-regulated under UV-B stress conditions in Arabidopsis (Zhou et al., 2007). Taken together, these data suggest that abiotic stresses have an important influence on miRNA expression. Drought is one of the most common environmental stresses that threaten agroforestry and result in deterioration of the environment. Given the fact that drought and salinity are becoming particularly widespread in many regions, it has been predicted that more than 50% of all arable land will be affected by serious salinization by the year 2050 (Wang et al., 2003; Boyer, 1982). Therefore, more effective xerophytic breeding strategies are urgently required to improve plant survival under extremely harsh environments. Novel drought-related miRNAs have been identified using novel methodologies such as highthroughput sequencing and oligonucleotide microarray in Arabidopsis and other model plants such as Oryza sativa, Populus euphratica, and
http://dx.doi.org/10.1016/j.gene.2015.07.072 0378-1119/© 2015 Published by Elsevier B.V.
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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Medicago truncatula. Thus, distinct responses to drought stresses were reported for miR393, miR397b, miR402, miR169a, and miR169c in Arabidopsis (Sunkar, 2001–2019; Li et al., 2008); miR170, miR172, miR397, miR408, miR529, and miR395 in O. sativa (Zhou et al., 2010); miR156a, miR162a, miR319a, miR396a, and miR166n in Populus (Li et al., 2011); and miR2118, miR399, miR164, miR169, miR171, miR396 in M. truncatula (Wang et al., 2011a). Previous studies have demonstrated that miR2118, which guides the mRNA cleavage of Toll interleukin-1 receptor–nucleotide-binding site– leucine-rich repeat (TIR–NBS–LRR) resistance genes, is closely related to drought response in legumes (Arenas-Huertero et al., 2009; Wang et al., 2011a; Kulcheski et al., 2011). For example, Arenas-Huertero et al. reported that pvu-miR2118 was responsive to drought and salinity in the common bean (Arenas-Huertero et al., 2009); Wang et al. found that miR2118 was up-regulated by drought stress in M. truncatula (Wang et al., 2011a); and Luo et al. identified that miR2118, which targets four TG01 genes, was strongly induced by drought stress (Luo, na). Caragana intermedia, of the Leguminosae family, is a deciduous, perennial shrub that is widespread in the sandy grassland and desert regions of northwest China and Mongolia (Wang et al., 2011b; Xu et al., 2007). It has important ecological and economic value in this area, including drought resistance, sand-fixation, water and soil conservation, as well as a nutritious pasture (Li et al., 2014; Xu et al., 2012). Because of its strong adaptability and stress tolerance, C. intermedia is an ideal candidate plant for studying the mechanisms of drought tolerance and creating drought-resistant forests in China. To date, however, only a few miRNAs have been identified in C. intermedia. 142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated in C. intermedia. Furthermore, the expression of 12 miRNAs was detected by qRT-PCR under salt stress, seven of which were upregulated, while one miRNA was downregulated (Zhu et al., 2013a). In this study, we predicted two potential target genes of miR2118 from the C. intermedia transcriptome and cloned them. We also characterized the expression patterns of miR2118 and its target genes under drought stress in various tissues of C. intermedia. To further this analysis, we generated transgenic Tobacco plants constitutively overexpressing cin-miR2118, and found that they showed enhanced drought stress tolerance. The results of this study contribute to a better understanding of the role of miR2118 and its target genes in drought stress responses.
2. Materials and methods 2.1. Plant materials and drought stress treatment C. intermedia seeds were washed three times in distilled water, soaked in distilled water for 6 h, then germinated in pots filled with sterilized soil in a growth chamber at 23 °C with a 16 h light/ 8 h dark photoperiod and relative humidity of 80%. Three-week-old seedlings were subjected to drought treatment by adding 15% polyethylene glycol (PEG) 6000 to 1/2 Hoagland's solution (Table S1) to mimic drought stress, then were sampled at 0, 2, 4, 8, 12, 24 and 48 h after treatment. The roots, stems, and leaves from 3-week-old seedlings and drought-stressed plants were harvested separately and stored at − 80 °C for RNA isolation. Samples for tissue-specific expression analysis were collected from three different seedlings as three biological replicates. Transgenic Tobacco drought tolerance assays were performed on the leaves of wild-type and transgenic seedlings of Nicotiana benthamiana. Seedlings were germinated on MS medium (Table S1) for 2 months, then the leaves were detached, placed at room temperature, and weighed on an electronic balance (Sartorius, Gottingen, Germany) every 20 min. Drought tolerance experiments were conducted in triplicate. The numerical data were subjected to statistical analysis using Excel 2010.
2.2. Full-length cDNA and sequence analysis We obtained sequence data for mature miR2118 and pre-miR2118 from the small RNA library of C. intermedia (unpublished results). Mature miR2118 target sequence prediction was performed with the online psRNATarget server using relatively strict rules (http://plantgrn. noble.org/psRNATarget/) including a maximum expectation value of 3. Total RNA was prepared from C. intermedia using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). First-strand cDNA was synthesized from total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). Rapid amplification of cDNA ends (RACE) was carried out to obtain 3′ and 5′ end cDNA sequences using the SMART™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA). RACE primers are described in Table S2. The open reading frame (ORF) was implemented using the NCBI ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). Domain searching was performed with the conserved domain database program (NCBI), and multiple alignments were obtained using Clustal X. Other bioinformatics analyses of this study, including the theoretical isoelectric point (pI), were predicted by the ExPASY bioinformatics resource portal (http:// web.expasy.org). 2.3. Quantification of gene expression (quantitative real-time PCR, qRT-PCR) RNA and total miRNA were extracted from the treated or untreated leaves, stems, and roots of 3-week-old plants. Total RNA isolation and first-strand cDNA synthesis were performed as described above. Total miRNA was extracted using an miRcute miRNA Isolation Kit (Tiangen Biotech), and first-strand cDNA synthesis of miRNA was conducted using an miRcute miRNA first-strand cDNA synthesis Kit (Tiangen Biotech). Transcript levels of the samples were detected by the CFX96™ realtime polymerase chain reaction (PCR) system (BioRad, Hercules, CA). The miRcute miRNA qPCR Detection Kit (SYBR Green) (Tiangen Biotech) was used for miRNA qRT-PCR with a forward primer designed according to the mature miR2118 sequence, and the reverse primer provided by the kit. C. intermedia 5.8S ribosomal RNA was selected as a reference gene (Table S2). Table S2 shows the CiDR1 and CiDR2 qRT-PCR primers for mRNA analysis. Expression levels of all target genes were normalized to that of UNK2 (Zhu et al., 2013b). SYBR Premix Ex Taq™ (Takara, Dalian, China) was used in the real-time PCR of target genes, and the set-up was according to the manufacturer's protocol. All reactions were repeated three times for each sample. Statistical analysis and sample comparisons were conducted using the 2−△△Ct method. 2.4. Plasmid constructs and the generation of transgenic Tobacco plants The overexpression construct was made by inserting a 256 bp premiR2118 fragment into the plant vector pSuper1300 + (Huayueyang, Beijing, China). The pre-miR2118 sequence including the fold-back structure was amplified from C. intermedia genomic DNA using the gene-specific primers cin-QTF and cin-QTR (Table S2). The plasmid containing the cDNA was double digested with Xba I and Kpn I, then the excised fragment was transfered into Trans-T1 competence-cell. The resultant reconstructed plasmid was introduced into the Agrobacterium strain GV3101 carrying the 35S promoter, then named 35S::preMIR2118. Transformation was achieved using an Agrobacteriummediated method as previously described (Dayan et al., 2010). 2.5. Anatomical observation For histological analysis, fresh stems from the same phase of 2month-old wild-type and transgenic tobacco plants were embedded in 3% agar. Approximately 20-μm-thick free-hand sections were cut
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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and stained with toluidine blue. The sections were observed and photographed under an AxioImager A1 microscope (Zeiss, Oberkochen, Germany) equipped with a computer-assisted AxioCam MRc5 camera (Zeiss). The pictures were further processed with Adobe Photoshop 7.
were induced by drought stress and helped plants adjust to the drought environment by regulating their expression.
3. Results
To further characterize the function of cin-miR2118, transgenic Tobacco plants overexpressing cin-miR2118 were generated. Two independent transgenic lines were selected for functional analysis (T-1, T-2). Cin-miR2118 expression in the roots, stems, and leaves of both transgenic Tobacco lines was upregulated compared with wild-type plants (WT) (Fig. 3A), indicating the stable expression of the transgene. To understand how cin-miR2118 affects the drought-resistant ability of Tobacco, we measured water loss from detached leaves and investigated the expression of cin-miR2118 in Tobacco. As shown in Fig. 3B, the detached leaves of transgenic lines (T-1, T-2) were observed to partially unfold, but those of wild-type plants (WT) were crinkled after 2 h drought treatment. Subsequent dehydration rate data more specifically illustrated this phenomenon. As shown in Fig. 3C, transgenic plants (T-1, T-2) lost water more quickly than wild-type (WT) during the first 60 min. After this time, the water loss rate of control plants quickly surpassed that of transgenic plants and remained substantially higher for the rest of the experiment. In light of the enhanced drought tolerance of miR2118-overexpressing plants, we examined whether miR2118 expression was associated with response to drought stress. As shown in Fig. 3D, miR2118 levels were greatly decreased after drought treatment, then increased slightly after 20 min and remained elevated for the remainder of the experiment. Because cin-miR2118 expression levels were consistent with water loss rate, these results indicated that miR2118 expression was induced by drought stress and that overexpression of cin-miR2118 increased drought tolerance in transgenic Tobacco plants.
3.1. Prediction, molecular cloning, and sequence analysis of cin-miR2118 target genes in C. intermedia miR2118-targeted genes have been identified in a diverse range of species, and most belong to the TIR–NBS–LRR gene family. To identify miR2118-targeted genes in C. intermedia, we searched for C. intermedia mRNA sequences containing complementary sequences to the mature miR2118 sequence. Two TIR–NBS–LRR genes were predicted to be putative targets, and designated CiDR1 (GenBank Accession No. KP277100) and CiDR2 (GenBank Accession No. KP744016). The cDNA sequences of CiDR1 and CiDR2 were 4297 bp and 3809 bp in length, including complete ORFs of 3732 bp and 3171 bp, respectively. The deduced protein lengths, molecular weights, and predicted pIs of the two target genes are shown in Table 1. Multiple-sequence alignments of full-length protein sequences revealed that both putative proteins contained the three highly conserved domains TIR, NBS, and LRR, suggesting that they are homologs of TIR– NBS–LRR resistance proteins (Fig. 1A). Further analysis showed that cin-miR2118 target sequences were present in the mRNA sequences of both genes within coding regions (Fig. 1B). 3.2. Drought-responsive expression patterns of mature cin-miR2118 and its target genes in C. intermedia To explore the possible involvement of CiDRs in the drought stress response, we first examined whether their expression could be induced by drought stress. We treated C. intermedia seedlings with 15% PEG and determined CiDR expression profiles in leaves, stems, and roots. After 48 h PEG treatment, growth was clearly repressed in the seedlings, and the older leaves had withered. The expression pattern of CiDRs in C. intermedia under drought stress was investigated using qRT-PCR. CiDR mRNAs were expressed at high levels in all organs of untreated plants. After treatment with 15% PEG for 2 h, however, CiDR1 transcription levels began to decrease in the leaves, stems, and roots. Although the transcription level rose slightly after 8 h, it immediately declined to a lower level than before (Fig. 2A). Similarly, CiDR2 expression was also modified in a timedependent manner by drought treatment. CiDR2 expression fell within 2 h of 15% PEG treatment, then recovered, peaked at 8 h (Fig. 2B), then repeated this cyclical variation. The expression pattern was similar among the roots, stems, and leaves. Based on these findings, we chose the stems as a representative organ to investigate the regulatory relationship between cin-miR2118 and its target genes. We used qRT-PCR to analyze transcript levels of cin-miR2118 and two of its targets to systematically validate whether they were regulated under drought stress. As shown in Fig. 2C, CiDR1 was negatively correlated with corresponding miR2118 expression at most phases under drought treatment, except for the 8 h time-point. By contrast, CiDR2 was positively correlated with cin-miR2118 at all time-points (Fig. 2D). Despite these differences in expression patterns, the results clearly indicated that cin-miR2118 and its target genes Table 1 Sequence analysis of miR2118 target genes in Caragana intermedia.
3.3. Overexpression of pre-miR2118 enhances drought tolerance in Tobacco
3.4. Overexpression of pre-miR2118 affected stem and root development in Tobacco Increasing the expression level of miR2118 drastically affected the normal development of stems and roots in Tobacco. All transgenic lines produced deformed organs, with shortened but incrassate roots and stronger stems. Compared with wild-type plants, the elongation of primary roots was much slower in transgenic lines, and it was difficult to identify taproots and lateral roots. Additionally, the lateral root primordia was observed closer to the stems of transgenic plants than in wild-type plants (Fig. 4A). These morphological alterations of transgenic lines suggested that miR2118 in Tobacco may be involved both in root growth and the generation of new root meristem. Almost 100% of roots from the transgenic lines showed changes in the maturation zone, characterized by the anomalous disappearance of root hairs (Fig. 4C). By contrast, the maturation zones of wild-type roots were very hairy (Fig. 4B). Root hair is a vital component of roots that expands the root contact area with the soil, enabling more moisture to be imbibed. We therefore speculated that transgenic lines would not survive after transfer into the soil because of the absence of root hairs. In the stems of transgenic Tobacco, no morphological alterations were observed in the cortex parenchyma, phloem, or cambium, whereas the overexpression of miR2118 increased both the number and size of xylem cells compared with wild-type (Fig. 4D, E). The layer number of xylem cells in wild type plants was only six in average, while there were 10 layers in transgenic lines, of which the xylem cells were more compact and orderly. 4. Discussion
Gene
ORF length
Deduced protein length (aa)
Deduced protein mol wt. (Da)
Deduced protein pI
CiDR1 CiDR2
3732 3171
1244 1057
141069.6 119903.9
6.35 6.46
Adversity stress is one of the conditions that can restrict crop growth and decrease production, so much research has attempted to understand this complex biological mechanism. Several 18–24 nt noncoding miRNAs have been shown to play a critical role in abiotic stress
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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Fig. 1. Bioinformatics analysis of cin-miR2118 target genes. (A) Predicted protein structure of CiDRs. Three domains (TIR, NB-ARC and LRR) are shown in different colors. (B) The alignment of miR2118 and CiDRs transcript sequences. Identical nucleotides between target sequences and mature miR2118 sequence are shown in red, the mismatched nucleotides are shown in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
responses through regulating their target gene expression by transcript cleavage, translation inhibition, or genomic DNA methylation (Bartel, 2004; Luan et al., 2015). The functional characterization of miRNAs relies heavily on the identification of their target genes (He and Hannon, 2004), which are recognized through base pairing, so computational methods have been invaluable (Jones-Rhoades and Bartel, 2004). Previous studies have reported that miR2118 is highly conserved in legume species and is involved in plant responses to a variety of abiotic stresses, especially drought (Arenas-Huertero et al., 2009; Jagadeeswaran et al., 2009; Johnson et al., 2009). In the present study, we predicted putative targets for cin-miR2118 using bioinformatics, and identified two genes homologous to known disease resistance genes with roles in plant defense (He et al., 2004; Shivaprasad et al., 2012). This prediction is consistent with work by Jagadeeswaran et al. in M. truncatula
(Jagadeeswaran et al., 2009). The two putative CiDR genes of this study contain three conserved domains as well as an miR2118 binding site (Fig. 1A,B). The LRR disease resistance protein TG01 was predicted to be the target of gma-miR2118 and in vivo cleavage by gma-miR2118 was identified by RLM-5′-RACE (Luo, nd). In the present study, we observed one or two mismatches between cin-miR2118 and target sites in CiDR sequences. These mismatches may affect the efficiency of miRNA cleavage. To date, a number of studies have identified miR2118 as a potential drought response gene in several plant species using high-throughput small RNA deep sequencing (Arenas-Huertero et al., 2009; Song et al., 2011). However, the functional verification of this is limited. We conducted functional studies of miR2118 and its target genes to determine whether they could be regulated by drought in C. intermedia. Quantitative reverse-transcription (qRT-PCR) showed that both CiDR1 and CiDR2
Fig. 2. Expression patterns of CiDRs in various tissues and regulation between cin-miR2118 and its target genes under drought stress for 2, 4, 8, 12, 24 and 48 h. (A–B) Expression patterns of CiDR1 and CiDR2 in different C. intermedia tissues under drought stress: (A) for CiDR1, (B) for CiDR2. (C–D) Regulation of cin-miR2118 and CiDR expression in stems under drought stress. Blue bars indicate the relative miR2118 levels, while yellow bars indicate their corresponding target CiDR1 expression and pink bars represent CiDR2 expression. Gene expression was normalized to the reference gene UNK2 (for CiDRs) and 5.8s-Caragana (for cin-miR2118). The data are averages of three replicates ± SE. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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Fig. 3. The effect of cin-miR2118 expression on osmotic stress in transgenic Tobacco plants. (A) Verification of cin-miR2118 overexpression in 35S::pre-MIR2118 transgenic Tobacco lines by quantitative RT-PCR. Different letters above each column indicate a significant difference (P b 0.05) according to Student's t test. (B) Water loss in detached leaves from 3-month plants placed in a room (40% rH) for 2 h. (C) Comparison of rates of water loss from detached leaves of the wild-type and two transgenic plants. Water loss is expressed as the proportion of initial fresh weight. Values are means ± SE from 10 leaves for each of three independent experiments. (D) Expression level of cin-miR2118 in transgenic Tobacco under drought for 20, 40, 60, 80, 100, 120 and 140 min. Gene expression was normalized to the reference gene 5.8s-Tobacco. The data are averages of three replicates ± SE. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. The effect of cin-miR2118 over-expressing on phenotype development in transgenic Tobacco plants. (A) Phenotypes of 2-month-old WT and transgenic seedlings grown in MS medium. Scale bar = 1 cm. (B–C) Histological study of WT and transgenic Tobacco roots stained with Evans blue. (B) Wild type. (C) Transgenic type. Scale bar = 100 μm. (D–E) Transverse sections of WT and 35S::miR2118 transgenic plant stems stained with toluidine blue. (D) Wild type. (E) Transgenic type. X means xylem tissues. Scale bar = 100 μm.
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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exhibited negative expression patterns under drought stress in C. intermedia seedlings (Fig. 2A,B), implicating their function in the plant response to drought stress. The expression pattern of cinmiR2118 and its target genes did not correspond well with the classical miRNA/target cascade model in which an miRNA transcript level increases and its target gene expression decreases. Instead, we observed an inverse relationship between CiDR1 transcript levels and cinmiR2118 abundance during drought stress (Fig. 2C), while CiDR2 and cin-miR2118 demonstrated a more consistent expression (Fig. 2D). It is conceivable that CiDR1 is the traditional target of miR2118, which is why its translation is repressed by complementary miR2118, and that miR2118 may trigger CiDR2 expression by inducing promoter expression as seen in an earlier study (Place et al., 2008). Alternatively, or additionally, the target gene CiDR2 may function as a “tuning targets” of miR2118, for which miRNA regulation adjusts the protein output in a fashion that permits customized expression in different cell types while achieving a more uniform expression level within each cell type (Williams et al., 2005; Bartel and Chen, 2004). CiDR1 and CiDR2 nevertheless appear to be potential targets of cin-miR2118 and to be strongly affected by drought stress. To further verify this, and because a transgenic system has not yet been developed in C. intermedia, we conducted functional experiments in Tobacco by overexpressing cin-miR2118 under the control of the 35S promoter. As shown in Fig. 3A, transgenic tobacco seedlings exhibited higher miR2118 expression in their roots, stems, and leaves than wild-type plants. The rate of water loss from detached leaves, as a measure of cuticular transpiration, has previously been suggested to be an important indicator of water status (Chen et al., 2006; Clarke et al., 1989). In our study, an unusual pattern of water loss occurred in that the rate from transgenic tobacco leaves exceeded that from wild-type leaves during the first 60 min, then the reverse pattern was observed (Fig. 3C). Subsequent quantitative RT-PCR analysis (Fig. 3D) revealed that cin-miR2118 expression greatly declined under drought stress, slowly recovered 20 min later, and peaked at 140 min. These results strongly indicate that the transgenic tobacco plants possessed a higher water retention ability than wild-type, and that cin-miR2118 functioned as a positive regulator in responding to drought stress. The current study also showed that overexpressing cin-miR2118 caused severe growth abnormalities in tobacco. cin-miR2118 overexpressing plants exhibited stronger stems, shorter but thicker roots, and an absence of root hairs in the maturation zone (Fig. 4B,C). Moreover, xylem cells were larger and more numerous than those in wildtype tobaccos, thus occupying more space between the central and peripheral zones (Fig. 4D,E). The xylem cells transport water and soluble mineral nutrients from the roots throughout the plant. They are also used to replace water lost during transpiration and photosynthesis. The increase of vessel number per unit area can provide guarantee for transportation system, improve the effectiveness and safety of water transportation, and also can reinforce the ability to bracing. This change in morphology exhibited distinctly xeromorphic characteristics. Factors influencing the formation of xylem structure are not only manifold, but involute. Although the relationship between overexpressed cinmiR2118 and the altered phenotype is unclear at the molecular level, we can assume that cin-miR2118 participates in the regulation of normal development in Tobacco. Additionally, to our knowledge, this is the first study showing the effect of miR2118 on plant growth. 5. Conclusions In conclusion, our experiments indicate that drought-induced cinmiR2118 expression down-regulates its CiDR1 target, which may encode a negative regulator of drought responses, but induces CiDR2 expression, using an uncertain molecular mechanism. Moreover, overexpressing cin-miR2118 in Tobacco led to an enhanced tolerance to drought stress. Our results therefore shed light on the possibility of manipulating miRNA to improve the tolerance of plants to drought stress.
Additionally, our assay revealed that cin-miR2118 may have a novel function as a growth regulator in Tobacco. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.07.072.
Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2011AA100203) and the National Natural Science Foundation of China (30830086).
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Gene journal homepage: www.elsevier.com/locate/gene
Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco Bi-fei Wu a, Wan-feng Li b, Hai-yan Xu b, Li-wang Qi b, Su-ying Han a,⁎ a b
Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
a r t i c l e
i n f o
Article history: Received 23 April 2015 Received in revised form 5 July 2015 Accepted 22 July 2015 Available online xxxx Keywords: Cin-miR2118 TIR-NBS-LRR Caragana intermedia Drought tolerance
a b s t r a c t The miR2118 is highly conserved in leguminous plants. Its function is to regulate the expression of genes encoding the TIR-NBS-LRR resistance protein. In this study, cin-miR2118 from Caragana intermedia was functionally characterized, especially with regard to its role in drought stress resistance. Two target genes of cin-miR2118 were predicted and cloned, the occurrence of miR2118 target sequence in both genes indicated that they might be targets of cin-miR2118. We investigated the expression patterns of cin-miR2118 and its target genes in C. intermedia stems and found diverse changes in expression in response to drought stress. CiDR1 was negatively correlated with corresponding miR2118 expression while CiDR2 was positively correlated with cin-miR2118. For further study, induced tolerance was observed in the transgenic Tobacco with overexpression cin-miR2118 upon 140-min water deficiency. And the expression level of cin-miR2118 was dramatically increased under drought stress. These results reveal that cin-miR2118 exert positive effects on drought stress tolerance. In addition, our study unexpectedly found that overexpression of cin-miR2118 in Tobacco can cause phenotype changes, which suggested that cin-miR2118 may have a novel function as a growth regulator in Tobacco. © 2015 Published by Elsevier B.V.
1. Introduction MicroRNAs (miRNAs) are ~ 21 nt endogenous RNAs that are highly conserved among higher plants (Chen and Rajewsky, 2007; Jones-Rhoades et al., 2006). They use base-pairing interactions to regulate mRNA expression (Bushati and Cohen, 2007) by cleaving the targeted transcript, or translational inhibition following imperfect pairing. Although both mechanisms have been observed in plants, cleavage is the more common (Arenas-Huertero et al., 2009; Brodersen et al., 2008). Numerous recent studies have shown that miRNAs and their targets play important roles in multiple biological processes, such as plant growth (Guo, 2005), organ development (Emery et al., 1768–1774; Kim et al., 2005), signal transduction (Wang et al., 2004), pathogen infection (Prokhnevsky and Chapman, 2004), and stress responses (Sunkar, 2001–2019). With the rapid development of molecular biology techniques, many novel miRNAs have been identified, although little is known about their functions or target genes (Sunkar, 2001–2019; He and Hannon, 2004). Some miRNAs have been confirmed experimentally to be involved in a variety of abiotic stress responses, encompassing drought, salinity,
Abbreviations: C. intermedia, Caragana intermedia; miRNA, microRNA; NCBI, National Center for Biotechnology Information; Pre, precursor; qRT-PCR, quantitative reverse transcription PCR; RLM-5′RACE, RNA ligase-mediated amplification of cDNA ends. ⁎ Corresponding author. E-mail address: [email protected] (S. Han).
extreme temperatures, nutrition stress, ultraviolet (UV)-B radiation, and oxidative stress. miR398 was the first miRNA reported to respond to abiotic stress. miR398-directed mRNA cleavage of target genes CSD1 and CSD2 fine-tunes their expression under oxidative stresses (Sunkar, 2006), while over-expressing osa-miR396c in rice and Arabidopsis reduced salt and alkali stress tolerance (Gao et al., 2010). Other studies found that miR399 is strongly induced by low phosphate stress, and that it controls phosphate homeostasis by regulating the expression of a putative ubiquitin-conjugating E2 enzyme, UBC24, in Arabidopsis (Fujii et al., 2005). miR169g and miR169n, which both target NF-YA, exhibited overlapping and distinct responses to drought and salt stresses (Zhao et al., 2009). Additionally, Zhou identified that 21 miRNA genes in 11 miRNA families are up-regulated under UV-B stress conditions in Arabidopsis (Zhou et al., 2007). Taken together, these data suggest that abiotic stresses have an important influence on miRNA expression. Drought is one of the most common environmental stresses that threaten agroforestry and result in deterioration of the environment. Given the fact that drought and salinity are becoming particularly widespread in many regions, it has been predicted that more than 50% of all arable land will be affected by serious salinization by the year 2050 (Wang et al., 2003; Boyer, 1982). Therefore, more effective xerophytic breeding strategies are urgently required to improve plant survival under extremely harsh environments. Novel drought-related miRNAs have been identified using novel methodologies such as highthroughput sequencing and oligonucleotide microarray in Arabidopsis and other model plants such as Oryza sativa, Populus euphratica, and
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Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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Medicago truncatula. Thus, distinct responses to drought stresses were reported for miR393, miR397b, miR402, miR169a, and miR169c in Arabidopsis (Sunkar, 2001–2019; Li et al., 2008); miR170, miR172, miR397, miR408, miR529, and miR395 in O. sativa (Zhou et al., 2010); miR156a, miR162a, miR319a, miR396a, and miR166n in Populus (Li et al., 2011); and miR2118, miR399, miR164, miR169, miR171, miR396 in M. truncatula (Wang et al., 2011a). Previous studies have demonstrated that miR2118, which guides the mRNA cleavage of Toll interleukin-1 receptor–nucleotide-binding site– leucine-rich repeat (TIR–NBS–LRR) resistance genes, is closely related to drought response in legumes (Arenas-Huertero et al., 2009; Wang et al., 2011a; Kulcheski et al., 2011). For example, Arenas-Huertero et al. reported that pvu-miR2118 was responsive to drought and salinity in the common bean (Arenas-Huertero et al., 2009); Wang et al. found that miR2118 was up-regulated by drought stress in M. truncatula (Wang et al., 2011a); and Luo et al. identified that miR2118, which targets four TG01 genes, was strongly induced by drought stress (Luo, na). Caragana intermedia, of the Leguminosae family, is a deciduous, perennial shrub that is widespread in the sandy grassland and desert regions of northwest China and Mongolia (Wang et al., 2011b; Xu et al., 2007). It has important ecological and economic value in this area, including drought resistance, sand-fixation, water and soil conservation, as well as a nutritious pasture (Li et al., 2014; Xu et al., 2012). Because of its strong adaptability and stress tolerance, C. intermedia is an ideal candidate plant for studying the mechanisms of drought tolerance and creating drought-resistant forests in China. To date, however, only a few miRNAs have been identified in C. intermedia. 142 miRNAs were identified and 38 miRNA targets were predicted, 4 of which were validated in C. intermedia. Furthermore, the expression of 12 miRNAs was detected by qRT-PCR under salt stress, seven of which were upregulated, while one miRNA was downregulated (Zhu et al., 2013a). In this study, we predicted two potential target genes of miR2118 from the C. intermedia transcriptome and cloned them. We also characterized the expression patterns of miR2118 and its target genes under drought stress in various tissues of C. intermedia. To further this analysis, we generated transgenic Tobacco plants constitutively overexpressing cin-miR2118, and found that they showed enhanced drought stress tolerance. The results of this study contribute to a better understanding of the role of miR2118 and its target genes in drought stress responses.
2. Materials and methods 2.1. Plant materials and drought stress treatment C. intermedia seeds were washed three times in distilled water, soaked in distilled water for 6 h, then germinated in pots filled with sterilized soil in a growth chamber at 23 °C with a 16 h light/ 8 h dark photoperiod and relative humidity of 80%. Three-week-old seedlings were subjected to drought treatment by adding 15% polyethylene glycol (PEG) 6000 to 1/2 Hoagland's solution (Table S1) to mimic drought stress, then were sampled at 0, 2, 4, 8, 12, 24 and 48 h after treatment. The roots, stems, and leaves from 3-week-old seedlings and drought-stressed plants were harvested separately and stored at − 80 °C for RNA isolation. Samples for tissue-specific expression analysis were collected from three different seedlings as three biological replicates. Transgenic Tobacco drought tolerance assays were performed on the leaves of wild-type and transgenic seedlings of Nicotiana benthamiana. Seedlings were germinated on MS medium (Table S1) for 2 months, then the leaves were detached, placed at room temperature, and weighed on an electronic balance (Sartorius, Gottingen, Germany) every 20 min. Drought tolerance experiments were conducted in triplicate. The numerical data were subjected to statistical analysis using Excel 2010.
2.2. Full-length cDNA and sequence analysis We obtained sequence data for mature miR2118 and pre-miR2118 from the small RNA library of C. intermedia (unpublished results). Mature miR2118 target sequence prediction was performed with the online psRNATarget server using relatively strict rules (http://plantgrn. noble.org/psRNATarget/) including a maximum expectation value of 3. Total RNA was prepared from C. intermedia using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China). First-strand cDNA was synthesized from total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). Rapid amplification of cDNA ends (RACE) was carried out to obtain 3′ and 5′ end cDNA sequences using the SMART™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA). RACE primers are described in Table S2. The open reading frame (ORF) was implemented using the NCBI ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). Domain searching was performed with the conserved domain database program (NCBI), and multiple alignments were obtained using Clustal X. Other bioinformatics analyses of this study, including the theoretical isoelectric point (pI), were predicted by the ExPASY bioinformatics resource portal (http:// web.expasy.org). 2.3. Quantification of gene expression (quantitative real-time PCR, qRT-PCR) RNA and total miRNA were extracted from the treated or untreated leaves, stems, and roots of 3-week-old plants. Total RNA isolation and first-strand cDNA synthesis were performed as described above. Total miRNA was extracted using an miRcute miRNA Isolation Kit (Tiangen Biotech), and first-strand cDNA synthesis of miRNA was conducted using an miRcute miRNA first-strand cDNA synthesis Kit (Tiangen Biotech). Transcript levels of the samples were detected by the CFX96™ realtime polymerase chain reaction (PCR) system (BioRad, Hercules, CA). The miRcute miRNA qPCR Detection Kit (SYBR Green) (Tiangen Biotech) was used for miRNA qRT-PCR with a forward primer designed according to the mature miR2118 sequence, and the reverse primer provided by the kit. C. intermedia 5.8S ribosomal RNA was selected as a reference gene (Table S2). Table S2 shows the CiDR1 and CiDR2 qRT-PCR primers for mRNA analysis. Expression levels of all target genes were normalized to that of UNK2 (Zhu et al., 2013b). SYBR Premix Ex Taq™ (Takara, Dalian, China) was used in the real-time PCR of target genes, and the set-up was according to the manufacturer's protocol. All reactions were repeated three times for each sample. Statistical analysis and sample comparisons were conducted using the 2−△△Ct method. 2.4. Plasmid constructs and the generation of transgenic Tobacco plants The overexpression construct was made by inserting a 256 bp premiR2118 fragment into the plant vector pSuper1300 + (Huayueyang, Beijing, China). The pre-miR2118 sequence including the fold-back structure was amplified from C. intermedia genomic DNA using the gene-specific primers cin-QTF and cin-QTR (Table S2). The plasmid containing the cDNA was double digested with Xba I and Kpn I, then the excised fragment was transfered into Trans-T1 competence-cell. The resultant reconstructed plasmid was introduced into the Agrobacterium strain GV3101 carrying the 35S promoter, then named 35S::preMIR2118. Transformation was achieved using an Agrobacteriummediated method as previously described (Dayan et al., 2010). 2.5. Anatomical observation For histological analysis, fresh stems from the same phase of 2month-old wild-type and transgenic tobacco plants were embedded in 3% agar. Approximately 20-μm-thick free-hand sections were cut
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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and stained with toluidine blue. The sections were observed and photographed under an AxioImager A1 microscope (Zeiss, Oberkochen, Germany) equipped with a computer-assisted AxioCam MRc5 camera (Zeiss). The pictures were further processed with Adobe Photoshop 7.
were induced by drought stress and helped plants adjust to the drought environment by regulating their expression.
3. Results
To further characterize the function of cin-miR2118, transgenic Tobacco plants overexpressing cin-miR2118 were generated. Two independent transgenic lines were selected for functional analysis (T-1, T-2). Cin-miR2118 expression in the roots, stems, and leaves of both transgenic Tobacco lines was upregulated compared with wild-type plants (WT) (Fig. 3A), indicating the stable expression of the transgene. To understand how cin-miR2118 affects the drought-resistant ability of Tobacco, we measured water loss from detached leaves and investigated the expression of cin-miR2118 in Tobacco. As shown in Fig. 3B, the detached leaves of transgenic lines (T-1, T-2) were observed to partially unfold, but those of wild-type plants (WT) were crinkled after 2 h drought treatment. Subsequent dehydration rate data more specifically illustrated this phenomenon. As shown in Fig. 3C, transgenic plants (T-1, T-2) lost water more quickly than wild-type (WT) during the first 60 min. After this time, the water loss rate of control plants quickly surpassed that of transgenic plants and remained substantially higher for the rest of the experiment. In light of the enhanced drought tolerance of miR2118-overexpressing plants, we examined whether miR2118 expression was associated with response to drought stress. As shown in Fig. 3D, miR2118 levels were greatly decreased after drought treatment, then increased slightly after 20 min and remained elevated for the remainder of the experiment. Because cin-miR2118 expression levels were consistent with water loss rate, these results indicated that miR2118 expression was induced by drought stress and that overexpression of cin-miR2118 increased drought tolerance in transgenic Tobacco plants.
3.1. Prediction, molecular cloning, and sequence analysis of cin-miR2118 target genes in C. intermedia miR2118-targeted genes have been identified in a diverse range of species, and most belong to the TIR–NBS–LRR gene family. To identify miR2118-targeted genes in C. intermedia, we searched for C. intermedia mRNA sequences containing complementary sequences to the mature miR2118 sequence. Two TIR–NBS–LRR genes were predicted to be putative targets, and designated CiDR1 (GenBank Accession No. KP277100) and CiDR2 (GenBank Accession No. KP744016). The cDNA sequences of CiDR1 and CiDR2 were 4297 bp and 3809 bp in length, including complete ORFs of 3732 bp and 3171 bp, respectively. The deduced protein lengths, molecular weights, and predicted pIs of the two target genes are shown in Table 1. Multiple-sequence alignments of full-length protein sequences revealed that both putative proteins contained the three highly conserved domains TIR, NBS, and LRR, suggesting that they are homologs of TIR– NBS–LRR resistance proteins (Fig. 1A). Further analysis showed that cin-miR2118 target sequences were present in the mRNA sequences of both genes within coding regions (Fig. 1B). 3.2. Drought-responsive expression patterns of mature cin-miR2118 and its target genes in C. intermedia To explore the possible involvement of CiDRs in the drought stress response, we first examined whether their expression could be induced by drought stress. We treated C. intermedia seedlings with 15% PEG and determined CiDR expression profiles in leaves, stems, and roots. After 48 h PEG treatment, growth was clearly repressed in the seedlings, and the older leaves had withered. The expression pattern of CiDRs in C. intermedia under drought stress was investigated using qRT-PCR. CiDR mRNAs were expressed at high levels in all organs of untreated plants. After treatment with 15% PEG for 2 h, however, CiDR1 transcription levels began to decrease in the leaves, stems, and roots. Although the transcription level rose slightly after 8 h, it immediately declined to a lower level than before (Fig. 2A). Similarly, CiDR2 expression was also modified in a timedependent manner by drought treatment. CiDR2 expression fell within 2 h of 15% PEG treatment, then recovered, peaked at 8 h (Fig. 2B), then repeated this cyclical variation. The expression pattern was similar among the roots, stems, and leaves. Based on these findings, we chose the stems as a representative organ to investigate the regulatory relationship between cin-miR2118 and its target genes. We used qRT-PCR to analyze transcript levels of cin-miR2118 and two of its targets to systematically validate whether they were regulated under drought stress. As shown in Fig. 2C, CiDR1 was negatively correlated with corresponding miR2118 expression at most phases under drought treatment, except for the 8 h time-point. By contrast, CiDR2 was positively correlated with cin-miR2118 at all time-points (Fig. 2D). Despite these differences in expression patterns, the results clearly indicated that cin-miR2118 and its target genes Table 1 Sequence analysis of miR2118 target genes in Caragana intermedia.
3.3. Overexpression of pre-miR2118 enhances drought tolerance in Tobacco
3.4. Overexpression of pre-miR2118 affected stem and root development in Tobacco Increasing the expression level of miR2118 drastically affected the normal development of stems and roots in Tobacco. All transgenic lines produced deformed organs, with shortened but incrassate roots and stronger stems. Compared with wild-type plants, the elongation of primary roots was much slower in transgenic lines, and it was difficult to identify taproots and lateral roots. Additionally, the lateral root primordia was observed closer to the stems of transgenic plants than in wild-type plants (Fig. 4A). These morphological alterations of transgenic lines suggested that miR2118 in Tobacco may be involved both in root growth and the generation of new root meristem. Almost 100% of roots from the transgenic lines showed changes in the maturation zone, characterized by the anomalous disappearance of root hairs (Fig. 4C). By contrast, the maturation zones of wild-type roots were very hairy (Fig. 4B). Root hair is a vital component of roots that expands the root contact area with the soil, enabling more moisture to be imbibed. We therefore speculated that transgenic lines would not survive after transfer into the soil because of the absence of root hairs. In the stems of transgenic Tobacco, no morphological alterations were observed in the cortex parenchyma, phloem, or cambium, whereas the overexpression of miR2118 increased both the number and size of xylem cells compared with wild-type (Fig. 4D, E). The layer number of xylem cells in wild type plants was only six in average, while there were 10 layers in transgenic lines, of which the xylem cells were more compact and orderly. 4. Discussion
Gene
ORF length
Deduced protein length (aa)
Deduced protein mol wt. (Da)
Deduced protein pI
CiDR1 CiDR2
3732 3171
1244 1057
141069.6 119903.9
6.35 6.46
Adversity stress is one of the conditions that can restrict crop growth and decrease production, so much research has attempted to understand this complex biological mechanism. Several 18–24 nt noncoding miRNAs have been shown to play a critical role in abiotic stress
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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Fig. 1. Bioinformatics analysis of cin-miR2118 target genes. (A) Predicted protein structure of CiDRs. Three domains (TIR, NB-ARC and LRR) are shown in different colors. (B) The alignment of miR2118 and CiDRs transcript sequences. Identical nucleotides between target sequences and mature miR2118 sequence are shown in red, the mismatched nucleotides are shown in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
responses through regulating their target gene expression by transcript cleavage, translation inhibition, or genomic DNA methylation (Bartel, 2004; Luan et al., 2015). The functional characterization of miRNAs relies heavily on the identification of their target genes (He and Hannon, 2004), which are recognized through base pairing, so computational methods have been invaluable (Jones-Rhoades and Bartel, 2004). Previous studies have reported that miR2118 is highly conserved in legume species and is involved in plant responses to a variety of abiotic stresses, especially drought (Arenas-Huertero et al., 2009; Jagadeeswaran et al., 2009; Johnson et al., 2009). In the present study, we predicted putative targets for cin-miR2118 using bioinformatics, and identified two genes homologous to known disease resistance genes with roles in plant defense (He et al., 2004; Shivaprasad et al., 2012). This prediction is consistent with work by Jagadeeswaran et al. in M. truncatula
(Jagadeeswaran et al., 2009). The two putative CiDR genes of this study contain three conserved domains as well as an miR2118 binding site (Fig. 1A,B). The LRR disease resistance protein TG01 was predicted to be the target of gma-miR2118 and in vivo cleavage by gma-miR2118 was identified by RLM-5′-RACE (Luo, nd). In the present study, we observed one or two mismatches between cin-miR2118 and target sites in CiDR sequences. These mismatches may affect the efficiency of miRNA cleavage. To date, a number of studies have identified miR2118 as a potential drought response gene in several plant species using high-throughput small RNA deep sequencing (Arenas-Huertero et al., 2009; Song et al., 2011). However, the functional verification of this is limited. We conducted functional studies of miR2118 and its target genes to determine whether they could be regulated by drought in C. intermedia. Quantitative reverse-transcription (qRT-PCR) showed that both CiDR1 and CiDR2
Fig. 2. Expression patterns of CiDRs in various tissues and regulation between cin-miR2118 and its target genes under drought stress for 2, 4, 8, 12, 24 and 48 h. (A–B) Expression patterns of CiDR1 and CiDR2 in different C. intermedia tissues under drought stress: (A) for CiDR1, (B) for CiDR2. (C–D) Regulation of cin-miR2118 and CiDR expression in stems under drought stress. Blue bars indicate the relative miR2118 levels, while yellow bars indicate their corresponding target CiDR1 expression and pink bars represent CiDR2 expression. Gene expression was normalized to the reference gene UNK2 (for CiDRs) and 5.8s-Caragana (for cin-miR2118). The data are averages of three replicates ± SE. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. The effect of cin-miR2118 expression on osmotic stress in transgenic Tobacco plants. (A) Verification of cin-miR2118 overexpression in 35S::pre-MIR2118 transgenic Tobacco lines by quantitative RT-PCR. Different letters above each column indicate a significant difference (P b 0.05) according to Student's t test. (B) Water loss in detached leaves from 3-month plants placed in a room (40% rH) for 2 h. (C) Comparison of rates of water loss from detached leaves of the wild-type and two transgenic plants. Water loss is expressed as the proportion of initial fresh weight. Values are means ± SE from 10 leaves for each of three independent experiments. (D) Expression level of cin-miR2118 in transgenic Tobacco under drought for 20, 40, 60, 80, 100, 120 and 140 min. Gene expression was normalized to the reference gene 5.8s-Tobacco. The data are averages of three replicates ± SE. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. The effect of cin-miR2118 over-expressing on phenotype development in transgenic Tobacco plants. (A) Phenotypes of 2-month-old WT and transgenic seedlings grown in MS medium. Scale bar = 1 cm. (B–C) Histological study of WT and transgenic Tobacco roots stained with Evans blue. (B) Wild type. (C) Transgenic type. Scale bar = 100 μm. (D–E) Transverse sections of WT and 35S::miR2118 transgenic plant stems stained with toluidine blue. (D) Wild type. (E) Transgenic type. X means xylem tissues. Scale bar = 100 μm.
Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072
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B. Wu et al. / Gene xxx (2015) xxx–xxx
exhibited negative expression patterns under drought stress in C. intermedia seedlings (Fig. 2A,B), implicating their function in the plant response to drought stress. The expression pattern of cinmiR2118 and its target genes did not correspond well with the classical miRNA/target cascade model in which an miRNA transcript level increases and its target gene expression decreases. Instead, we observed an inverse relationship between CiDR1 transcript levels and cinmiR2118 abundance during drought stress (Fig. 2C), while CiDR2 and cin-miR2118 demonstrated a more consistent expression (Fig. 2D). It is conceivable that CiDR1 is the traditional target of miR2118, which is why its translation is repressed by complementary miR2118, and that miR2118 may trigger CiDR2 expression by inducing promoter expression as seen in an earlier study (Place et al., 2008). Alternatively, or additionally, the target gene CiDR2 may function as a “tuning targets” of miR2118, for which miRNA regulation adjusts the protein output in a fashion that permits customized expression in different cell types while achieving a more uniform expression level within each cell type (Williams et al., 2005; Bartel and Chen, 2004). CiDR1 and CiDR2 nevertheless appear to be potential targets of cin-miR2118 and to be strongly affected by drought stress. To further verify this, and because a transgenic system has not yet been developed in C. intermedia, we conducted functional experiments in Tobacco by overexpressing cin-miR2118 under the control of the 35S promoter. As shown in Fig. 3A, transgenic tobacco seedlings exhibited higher miR2118 expression in their roots, stems, and leaves than wild-type plants. The rate of water loss from detached leaves, as a measure of cuticular transpiration, has previously been suggested to be an important indicator of water status (Chen et al., 2006; Clarke et al., 1989). In our study, an unusual pattern of water loss occurred in that the rate from transgenic tobacco leaves exceeded that from wild-type leaves during the first 60 min, then the reverse pattern was observed (Fig. 3C). Subsequent quantitative RT-PCR analysis (Fig. 3D) revealed that cin-miR2118 expression greatly declined under drought stress, slowly recovered 20 min later, and peaked at 140 min. These results strongly indicate that the transgenic tobacco plants possessed a higher water retention ability than wild-type, and that cin-miR2118 functioned as a positive regulator in responding to drought stress. The current study also showed that overexpressing cin-miR2118 caused severe growth abnormalities in tobacco. cin-miR2118 overexpressing plants exhibited stronger stems, shorter but thicker roots, and an absence of root hairs in the maturation zone (Fig. 4B,C). Moreover, xylem cells were larger and more numerous than those in wildtype tobaccos, thus occupying more space between the central and peripheral zones (Fig. 4D,E). The xylem cells transport water and soluble mineral nutrients from the roots throughout the plant. They are also used to replace water lost during transpiration and photosynthesis. The increase of vessel number per unit area can provide guarantee for transportation system, improve the effectiveness and safety of water transportation, and also can reinforce the ability to bracing. This change in morphology exhibited distinctly xeromorphic characteristics. Factors influencing the formation of xylem structure are not only manifold, but involute. Although the relationship between overexpressed cinmiR2118 and the altered phenotype is unclear at the molecular level, we can assume that cin-miR2118 participates in the regulation of normal development in Tobacco. Additionally, to our knowledge, this is the first study showing the effect of miR2118 on plant growth. 5. Conclusions In conclusion, our experiments indicate that drought-induced cinmiR2118 expression down-regulates its CiDR1 target, which may encode a negative regulator of drought responses, but induces CiDR2 expression, using an uncertain molecular mechanism. Moreover, overexpressing cin-miR2118 in Tobacco led to an enhanced tolerance to drought stress. Our results therefore shed light on the possibility of manipulating miRNA to improve the tolerance of plants to drought stress.
Additionally, our assay revealed that cin-miR2118 may have a novel function as a growth regulator in Tobacco. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.07.072.
Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2011AA100203) and the National Natural Science Foundation of China (30830086).
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Please cite this article as: Wu, B., et al., Role of cin-miR2118 in drought stress responses in Caragana intermedia and Tobacco, Gene (2015), http:// dx.doi.org/10.1016/j.gene.2015.07.072